US20140323327A1 - Animals, repertoires & methods - Google Patents

Animals, repertoires & methods Download PDF

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US20140323327A1
US20140323327A1 US14/220,099 US201414220099A US2014323327A1 US 20140323327 A1 US20140323327 A1 US 20140323327A1 US 201414220099 A US201414220099 A US 201414220099A US 2014323327 A1 US2014323327 A1 US 2014323327A1
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human
repertoire
gene
vertebrates
gene segments
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Allan Bradley
E-Chiang Lee
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Kymab Ltd
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Kymab Ltd
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Priority claimed from GB201116122A external-priority patent/GB201116122D0/en
Priority claimed from GB201116120A external-priority patent/GB201116120D0/en
Priority claimed from GBGB1203257.9A external-priority patent/GB201203257D0/en
Priority claimed from GBGB1204592.8A external-priority patent/GB201204592D0/en
Priority claimed from GBGB1205702.2A external-priority patent/GB201205702D0/en
Priority claimed from GB1207814.3A external-priority patent/GB2501753A/en
Priority claimed from GBGB1208749.0A external-priority patent/GB201208749D0/en
Priority claimed from GB201211692A external-priority patent/GB201211692D0/en
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Assigned to KYMAB LIMITED reassignment KYMAB LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BRADLEY, ALLAN, LEE, E-CHIANG
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/204—Pseudochromosomes, minichrosomosomes of bacterial origin, e.g. BAC

Definitions

  • the present invention is directed to the concept of sectoring antibody gene segment repertoires in order to enable the development of novel, synthetic antibody chain repertoires not seen in nature.
  • Sectoring exploits the finite B-cell compartments of non-human vertebrates (such as mice and rats) by artificially biasing the antibody gene segment repertoire available for the production of antibody sequences in the B-cell compartments of individual na ⁇ ve and immunised vertebrates.
  • a plurality of these vertebrates together are useful as a population in immunisation schedules and research programmes to provide for access to a combined, synthetic antibody gene segment repertoire that is beyond that seen in nature and in prior art transgenic vertebrates in which antibody loci have been engineered.
  • the present invention is also directed to the realisation of the inventors that sectoring can also alter gene segment expression by providing new arrangements of gene segment clusters relative to other gene segments and regulatory elements in transgenic immunoglobulin loci, thereby providing for new synthetic antibody chain sequence repertoires.
  • the present invention provides novel, synthetically-extended antibody repertoires and immunoglobulin heavy and light chain sequence repertoires in non-human vertebrates.
  • the present invention also provides methods of selecting an antibody from said repertoires as well as populations of non-human vertebrates (such as mice or rats) that together provide the novel synthetic (non-naturally occurring) repertoires.
  • the invention also provides for particular non-human vertebrates that are biased to human lambda variable region expression substantially in the absence of kappa chain expression. Such vertebrates are useful in sectoring the kappa and lambda V gene repertoires to provide for novel light chain sequence repertoires according to the invention.
  • the invention also relates to inversion of vertebrate gene segments and use of these to construct transgenic antibody chain loci in which the inverted gene segments are functional and can contribute to new, synthetic, antibody chain and variable region repertoires.
  • the state of the art provides non-human vertebrates (eg, mice and rats) and cells comprising transgenic immunoglobulin loci, such loci comprising human variable (V), diversity (D) and/or joining (J) segments, and optionally human constant regions.
  • transgenic immunoglobulin loci such loci comprising human variable (V), diversity (D) and/or joining (J) segments, and optionally human constant regions.
  • endogenous constant regions of the host vertebrate eg, mouse or rat constant regions
  • Methods of constructing such transgenic vertebrates and use of these to generate antibodies and nucleic acids thereof following antigen immunisation are known in the art, eg, see U.S. Pat. No. 7,501,552 (Medarex); U.S. Pat. No. 5,939,598 & U.S. Pat. No.
  • transgenic non-human vertebrates It would be desirable to improve upon the prior art transgenic non-human vertebrates to provide for novel and potentially expanded repertoires and diversity of antibodies in na ⁇ ve and immunised non-human vertebrates bearing transgenic immunoglobulin loci.
  • the present inventors addressed this by devising ways of sectoring antibody gene segment repertoires by dividing the repertoire across members of a population of antibody-generating non-human vertebrates.
  • the inventors realised that the potential for accessing greater gene segment sequence diversity (eg, human antibody gene segment and human antibody variable region diversity) would be made possible by biasing B-cell compartments of individual vertebrates to restricted gene segment sub-repertoires, so that overall the population of vertebrates enables one to access novel gene segment (and resultant antibody sequence) repertoires and potentially explore extended ranges of antibody diversities that are not produced by the prior art transgenic vertebrate collections.
  • distal VH gene segment cluster eg, 5 to 10 distal human germline VH gene segments
  • D and JH genes eg, a substantially complete human D and JH repertoire
  • the invention provides the following.
  • a method of providing a synthetic antibody heavy chain sequence repertoire comprising providing a heavy chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous heavy chain expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic heavy chain loci comprising one or more VH gene segments, one or more D gene segments and one or JH gene segments functionally connected upstream of a heavy chain constant region (eg, Cmu and/or Cgamma), wherein the genomes can express different repertoires of antibody heavy chain sequences derived from VH, D and JH gene segments;
  • a heavy chain constant region eg, Cmu and/or Cgamma
  • the gene segment repertoire is selected from the group consisting of: (a) a VH gene repertoire (eg, a human VH gene repertoire or a substantially complete functional human VH gene repertoire); (b) a D gene repertoire (eg, a human D gene repertoire or a substantially complete functional human D gene repertoire); and (c) a JH gene repertoire (eg, a human JH gene repertoire or a substantially complete functional human JH gene repertoire); Optionally wherein the D and JH segments in the loci are human D and JH segments.
  • a VH gene repertoire eg, a human VH gene repertoire or a substantially complete functional human VH gene repertoire
  • D gene repertoire eg, a human D gene repertoire or a substantially complete functional human D gene repertoire
  • a JH gene repertoire eg, a human JH gene repertoire or a substantially complete functional human JH gene repertoire
  • a method of providing a synthetic antibody kappa chain sequence repertoire comprising providing a kappa chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic light chain loci comprising one or more V ⁇ gene segments and one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody kappa chain sequences derived from V ⁇ and J ⁇ gene segments;
  • a light chain constant region eg, C ⁇ and/or C ⁇
  • the gene segment repertoire is selected from the group consisting of: (a) a V ⁇ gene repertoire (eg, a human V ⁇ gene repertoire or a substantially complete functional human V ⁇ gene repertoire); and (c) a J ⁇ gene repertoire (eg, a human J ⁇ gene repertoire or a substantially complete functional human J ⁇ gene repertoire); Optionally wherein the J ⁇ segments in the loci are human J ⁇ segments.
  • a method of providing a synthetic antibody lambda chain sequence repertoire comprising providing a lambda chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic light chain loci comprising one or more V ⁇ gene segments and one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody lambda chain sequences derived from V ⁇ and J ⁇ gene segments;
  • a light chain constant region eg, C ⁇ and/or C ⁇
  • the gene segment repertoire is selected from the group consisting of: (a) a V ⁇ gene repertoire (eg, a human V ⁇ gene repertoire or a substantially complete functional human V ⁇ gene repertoire); and (c) a J ⁇ gene repertoire (eg, a human J ⁇ gene repertoire or a substantially complete functional human J ⁇ gene repertoire); Optionally wherein the J ⁇ segments in the loci are human J ⁇ segments.
  • a method of providing a synthetic antibody light chain sequence repertoire comprising providing
  • V ⁇ gene repertoire in the genomes of a first group of non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ , eg, human C ⁇ ), wherein the genomes can express repertoires of antibody kappa light chain sequences derived from V ⁇ and J ⁇ gene segments substantially in the absence of lambda light chain expression; and a V ⁇ gene repertoire in the genomes of a second group of non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇
  • a method of providing a synthetic antibody heavy chain sequence repertoire comprising providing a VH gene repertoire (eg, a substantially complete functional human VH gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous heavy chain expression is substantially inactive, the VH genes in the genomes being provided as part of transgenic heavy chain loci comprising one or more human JH gene segments and one or more human D gene segments functionally connected upstream of a heavy chain constant region (eg, Cmu and/or Cgamma), wherein the genomes can express different repertoires of antibody heavy chain sequences derived from human VH, D and JH gene segments.
  • the VH gene repertoire is a human VH gene repertoire.
  • a method of providing a synthetic antibody heavy chain sequence repertoire comprising providing a JH gene repertoire (eg, a substantially complete functional human JH gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous heavy chain expression is substantially inactive, the JH genes in the genomes being provided as part of transgenic heavy chain loci comprising one or more human VH gene segments and one or more human D gene segments functionally connected upstream of a heavy chain constant region (eg, Cmu and/or Cgamma), wherein the genomes can express different repertoires of antibody heavy chain sequences derived from human VH, D and JH gene segments.
  • the JH gene repertoire is a human JH gene repertoire.
  • a method of providing a synthetic antibody heavy chain sequence repertoire comprising providing a D gene repertoire (eg, a substantially complete functional human JH gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous heavy chain expression is substantially inactive, the D genes in the genomes being provided as part of transgenic heavy chain loci comprising one or more human VH gene segments and one or more human JH gene segments functionally connected upstream of a heavy chain constant region (eg, Cmu and/or Cgamma), wherein the genomes can express different repertoires of antibody heavy chain sequences derived from human VH, D and JH gene segments.
  • the D gene repertoire is a human D gene repertoire.
  • a method of providing a synthetic antibody kappa light chain sequence repertoire comprising providing a V ⁇ gene repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more human J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody kappa light chain sequences derived from human V ⁇ and J ⁇ gene segments.
  • the V ⁇ gene repertoire is a human V ⁇ gene repertoire.
  • a method of providing a synthetic antibody kappa light chain sequence repertoire comprising providing a J ⁇ gene repertoire (eg, a substantially complete functional human J ⁇ gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the J ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more human V ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody kappa light chain sequences derived from human V ⁇ and J ⁇ gene segments.
  • the J ⁇ gene repertoire is a human J ⁇ gene repertoire.
  • a method of providing a synthetic antibody lambda light chain sequence repertoire comprising providing a V ⁇ gene repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive, the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more human J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ , eg, human C ⁇ ), wherein the genomes can express different repertoires of antibody lambda light chain sequences derived from human V ⁇ and J ⁇ gene segments.
  • the V ⁇ gene repertoire is a human V ⁇ gene repertoire.
  • a method of providing a synthetic antibody lambda light chain sequence repertoire comprising providing J ⁇ gene repertoire (eg, a substantially complete functional human J ⁇ gene repertoire) that is divided across the genomes of two or more non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive, the J ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more human V ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ , eg, human C ⁇ ), wherein the genomes can express different repertoires of antibody lambda light chain sequences derived from human V ⁇ and J ⁇ gene segments.
  • the J ⁇ gene repertoire is a human J ⁇ gene repertoire.
  • a method of providing a synthetic antibody light chain sequence repertoire comprising providing
  • V ⁇ gene repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) in the genomes of a first group of non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive
  • the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more human J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ , eg, human C ⁇ ), wherein the genomes can express repertoires of antibody kappa light chain sequences derived from human V ⁇ and J ⁇ gene segments substantially in the absence of kappa light chain expression
  • a V ⁇ gene repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) in the genomes of a second group of non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive
  • the V ⁇ genes in the genomes being provided as part of transgenic light chain
  • a method of providing a synthetic antibody heavy chain sequence repertoire comprising
  • a method of providing a synthetic antibody light chain sequence repertoire comprising
  • a method of selecting an antibody that binds a predetermined antigen comprising
  • a population of transgenic non-human vertebrates (optionally mice or rats), wherein the population provides a repertoire of different human VH gene segments, the repertoire being divided between two or more vertebrates of said population,
  • first vertebrate of said population comprising a transgenic heavy chain locus comprising one or more human VH gene segments (first VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; and (b) a second vertebrate of said population comprising a transgenic heavy chain locus comprising one or more human VH gene segments (second VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; (c) wherein the first VH gene sub-repertoire is different from the second VH gene sub-repertoire for expression of first and second antibody heavy chain sequence repertoires respectively that are different from each other, whereby the population provides a synthetic repertoire of antibody heavy chain sequences.
  • a population of transgenic non-human vertebrates (optionally mice or rats), wherein the population provides a repertoire of different human VL gene segments, the repertoire being divided between two or more vertebrates of said population,
  • first vertebrate of said population comprising a transgenic light chain locus comprising one or more human VL gene segments (first VL gene sub-repertoire) and J segments operably connected upstream of a constant region; and (b) a second vertebrate of said population comprising a transgenic light chain locus comprising one or more human VL gene segments (second VL gene sub-repertoire) and J segments operably connected upstream of a constant region; (c) wherein the first VL gene sub-repertoire is different from the second VL gene sub-repertoire for expression of first and second antibody light chain sequence repertoires respectively that are different from each other, whereby the population provides a synthetic repertoire of antibody light chain sequences.
  • a population of non-human vertebrates (optionally mice or rats), wherein the genome of each vertebrate comprises:
  • transgenic immunoglobulin heavy chain loci each locus comprising one or more human V gene segments, one or more human D gene segments and one or more human J gene segments upstream of one or more heavy chain constant regions
  • transgenic immunoglobulin light chain loci each locus comprising one or more human V L gene segments and one or more human J L gene segments upstream of one or more light chain constant regions
  • the gene segments in transgenic heavy chain loci are operably linked to the constant region thereof, and the gene segments in transgenic light chain loci are operably linked to the constant region thereof, so that upon immunisation the vertebrate is capable of producing an antibody comprising heavy chains produced by recombination of a heavy chain locus and light chains produced by recombination of a light chain locus, wherein the heavy and light chains comprise human variable regions
  • the population comprises (i) a first vertebrate type (lambda vertebrates) wherein said light chain loci comprise one or
  • a non-human vertebrate (optionally a mouse or rat), wherein the genome of each vertebrate comprises:
  • transgenic immunoglobulin heavy chain loci each locus comprising one or more human V gene segments, one or more human D gene segments and one or more human J gene segments upstream of one or more heavy chain constant regions; and (d) One or more transgenic immunoglobulin light chain loci, each locus comprising a human V ⁇ gene segment repertoire and one or more human J ⁇ gene segments upstream of one or more light chain constant regions; Wherein following rearrangement the light chain loci express light chain sequences comprising variable region sequences derived from human V ⁇ gene segments (human lambda light chain sequences); Wherein the kappa (and optionally endogenous lambda) light chain expression has been substantially inactivated so that the vertebrate expresses more human lambda light chain sequences than kappa light chain sequences (sequences of light chains comprising variable region sequences derived from V ⁇ gene segments); Wherein endogenous heavy chain expression has been substantially inactivated; and Wherein each said transgenic light chain locus comprises a substantially
  • a method of providing a synthetic antibody heavy chain repertoire comprising
  • a human VH gene segment repertoire (eg, a substantially complete functional human VH gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of VH gene segments corresponding to a distal VH gene cluster of the heavy chain locus of a human; and a second cluster of VH gene segments corresponding to a proximal VH gene cluster of the heavy chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a heavy chain locus of said first vertebrate upstream of one or more D gene segments, one or more JH gene segments and one or more constant regions; Wherein the proximal cluster is provided in a heavy chain locus of said second vertebrate upstream of one or more D gene segments, one or more JH gene segments and one or more constant regions; Wherein the proximal VH gene cluster is not present between
  • a method of providing a synthetic antibody kappa chain repertoire comprising
  • a human V ⁇ gene segment repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of V ⁇ gene segments corresponding to a distal V ⁇ gene cluster of the kappa chain locus of a human; and a second cluster of V ⁇ gene segments corresponding to a proximal V ⁇ gene cluster of the kappa chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a kappa chain locus of said first vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal cluster is provided in a kappa chain locus of said second vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal V ⁇ gene cluster is not present between the distal cluster and a
  • a method of providing a synthetic antibody lambda chain repertoire comprising
  • a human V ⁇ gene segment repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of V ⁇ gene segments corresponding to a distal V ⁇ gene cluster of the lambda chain locus of a human; and a second cluster of V ⁇ gene segments corresponding to a proximal V ⁇ gene cluster of the lambda chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a lambda chain locus of said first vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal cluster is provided in a lambda chain locus of said second vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal V ⁇ gene cluster is not present between the distal cluster and the
  • a non-human vertebrate (optionally a mouse or a rat) or vertebrate cell (optionally a mouse cell or a rat cell) whose genome comprises a transgenic antibody chain locus comprising one or more human V gene segments and one or more human J gene segments (and optionally one or more human D gene segments) upstream of a constant region, the locus comprising one or more inverted vertebrate species gene segments, the inverted gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the vertebrate species germline orientation of such segment(s), and wherein the non-human vertebrate or cell is capable of expressing an antibody chain sequence comprising a sequence that is derived from an inverted gene segment.
  • the gene segments are naturally present in an opposite orientation to the corresponding CL or C ⁇ in the germline locus of a vertebrate of the relevant species (eg, human), but by virtue of the present invention these are inverted so that the gene segment orientation is the same as the CL or C ⁇ .
  • a non-human vertebrate (optionally a mouse or a rat) or vertebrate cell (optionally a mouse cell or a rat cell) whose genome comprises a transgenic antibody chain locus comprising one or more human V gene segments and one or more human J gene segments (and optionally one or more human D gene segments) upstream of a constant region, wherein the locus comprises one or more inverted human gene segments, the inverted human gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the human germline orientation of such segment(s), and wherein the vertebrate or cell is capable of expressing an antibody chain sequence comprising a variable region that is derived from recombination of an inverted gene segment.
  • a method of providing an artificial human antibody variable region repertoire comprising inserting one or more human V gene segment(s) (inverted gene segments) upstream of one or more J gene segments, optionally one or more D gene segments, and a constant region in an antibody chain locus of a non-human vertebrate or non-human vertebrate cell, the V gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the human germline orientation of such segment(s), and wherein the non-human vertebrate or cell (or a non-human vertebrate progeny derived from the cell) is capable of expressing an antibody chain sequence comprising a variable region sequence that is derived from recombination of an inverted gene segment.
  • a method of providing an artificial human antibody variable region repertoire comprising isolating serum or lymphoid cells (eg, spleen cells or B-cells) from a vertebrate described above, and optionally isolating from the serum or cells one or more antibodies that specifically bind a predetermined antigen.
  • serum or lymphoid cells eg, spleen cells or B-cells
  • FIG. 1 Schematic showing calculation of potential IgH antibody variable region diversity for a transgenic human immunoglobulin heavy chain locus containing 6 human JH gene segments, 27 human D gene segments and 40 VH gene segments.
  • FIG. 2 Schematic showing calculation of potential Ig ⁇ and Ig ⁇ antibody variable region diversity for transgenic human immunoglobulin light chain kappa and lambda loci, wherein each light chain locus contains 5 human J L gene segments and 40 V L gene segments.
  • FIG. 3 Schematic showing calculation of potential IgH, Ig ⁇ and Ig ⁇ antibody variable region diversity for the transgenic human immunoglobulin loci, giving a total potential antibody repertoire size of 4 ⁇ 10 14 .
  • FIG. 4 Schematic showing the typical sampling size in prior art methods of antibody selection from transgenic mice and the typical resultant hybridoma population size.
  • FIG. 5 Schematic showing sectoring of the light chain diversity across several mice in a population according to the invention and the resultant beneficial new and extended repertoire for sampling.
  • FIG. 6 Schematic showing sectoring of human functional light chain diversity and human functional VH gene segment diversity across several mice in a population according to the invention and the resultant beneficial new and extended repertoire for sampling.
  • FIG. 7 shows the human gene segment repertoires contained in the first, second and third BACs used to construct three different mice lines, K1, K2 and K3:—
  • K1 comprises an endogenous Ig kappa locus in which has been inserted the human gene segments V ⁇ 1-19 to J ⁇ 5
  • K2 comprises an endogenous Ig kappa locus in which has been inserted the human gene segments V ⁇ 2-24 to J ⁇ 5
  • K3 comprises an endogenous Ig kappa locus in which has been inserted the human gene segments V ⁇ 2D-40 to J ⁇ 5.
  • FIG. 8 shows total human V ⁇ gene segment usage versus mouse V ⁇ usage in transcripts from mice comprising an insertion of human V ⁇ and J ⁇ gene segments into an endogenous mouse kappa locus between the 3′-most mouse J ⁇ and the mouse C ⁇ .
  • K3/KA K3—the first endogenous kappa allele has three kappa chain locus DNA insertions between the most 3′ endogenous J ⁇ and the mouse CK, providing an insertion of 20 human V ⁇ and J ⁇ 1-J ⁇ 5; and (ii) KA—the second endogenous kappa allele has been inactivated (by insertion of an endogenous interrupting sequence).
  • This arrangement encodes exclusively for kappa light chains from the first endogenous kappa allele.
  • FIG. 9 illustrates the improvement in human V ⁇ gene segment usage in transcripts (and thus improvement in inactivation of endogenous mouse V ⁇ gene segment usage) as human kappa locus DNA is inserted in successive BACs 1-3.
  • BAC1 inserts 6 human V ⁇ and J ⁇ 1-J ⁇ 5 between the 3′ endogenous J ⁇ and the mouse C ⁇ .
  • BAC2 inserts an additional 8 human V ⁇ gene segments 5′ to the human DNA inserted from the BAC1, resulting in 14 human V ⁇ and J ⁇ 1-J ⁇ 5 between the 3′ endogenous J ⁇ and the mouse C ⁇ .
  • BAC3 inserts an additional 6 human V ⁇ gene segments 5′ to the human DNA inserted from the BAC2, resulting in 20 human V ⁇ and J ⁇ 1-J ⁇ 5 between the 3′ endogenous J ⁇ and the mouse C ⁇ . With each insertion, the endogenous mouse VJ region is pushed further upstream (5′) and inactivation is enhanced.
  • FIG. 10 illustrates the distribution of human V ⁇ usage at the transcript level from the K3/KA mice.
  • FIG. 11 compares the effect of sectoring the 20 human V ⁇ gene segment repertoire across three different mice-types (corresponding to insertion of human gene segment DNA from BAC1 only (K1 genotype), BACs1+2 (K2 genotype) and BACs1+2+3 (K3 genotype). Illustrated is the distribution of human V ⁇ usage at the transcript level from the mice. Different V ⁇ usage was seen resulting in mice that produced different kappa chain repertoires (and corresponding different human kappa variable region repertoires) as a result of the human gene repertoire sectoring.
  • FIG. 12 compares the effect of sectoring the human J ⁇ gene segment repertoire across three different mice-types (K1, K2 and K3). Illustrated is the distribution of human J ⁇ usage at the transcript level from the mice. Different J ⁇ usage was seen resulting in mice that produced different kappa chain repertoires (and corresponding different human kappa variable region repertoires) as a result of the human gene repertoire sectoring.
  • FIG. 13 illustrates the improvement in human VH gene segment usage in transcripts (and thus improvement in inactivation of endogenous mouse VH gene segment usage) as human heavy chain locus DNA is inserted in successive BACs 1-3 (producing S1 chimaeric heavy chain locus (human VDJ gene segments from BAC1 only have been inserted), S2 chimaeric heavy chain locus (human VDJ gene segments from BACs1 & 2 have been inserted) and S3 chimaeric heavy chain locus ((human VDJ gene segments from BACs 1, 2 & 3 have been inserted)).
  • BAC1 inserts 6 human VH and all functional human DH and JH gene segments between the 3′ endogenous JH (mouse JH4) and the mouse C-mu.
  • BAC2 inserts an additional 5 human VH gene segments 5′ to the human DNA inserted from the BAC1, resulting in 11 human VH and all functional human DH and JH between the 3′ endogenous JH4 and the mouse C-mu.
  • BAC3 inserts an additional 7 human VH gene segments 5′ to the human DNA inserted from the BAC2, resulting in 18 human VH and all functional human DH and JH between the 3′ endogenous JH4 and the mouse C-mu. With each insertion, the endogenous mouse heavy chain VDJ region is pushed further upstream (5′) and inactivation is enhanced.
  • BAC1 human gene segments VH2-5, 7-4-1, 4-4, 1-3, 1-2, 6-1, and all the human D and JH gene segments D1-1, 2-2, 3-9, 3-10, 4-11, 5-12, 6-13, 1-14, 2-15, 3-16, 4-17, 5-18, 6-19, 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 1-26 and 7-27; and J1, J2, J3, J4, J5 and J6.
  • BAC2 human gene segments VH3-7, 1-8, 3-9, 3-11 and 3-13.
  • BAC3 human gene segments VH3-15, 1-18, 3-20, 3-21, 3-23, 1-24 and 2-26.
  • FIG. 14 compares the effect of sectoring the 18 human VH gene segment repertoire across three different mice-types (corresponding to insertion of human gene segment DNA from BAC1 only (S1 genotype), BACs1+2 (S2 genotype) and BACs1+2+3 (S3 genotype). Illustrated is the distribution of human VH usage at the transcript level from the mice. Different VH usage was seen resulting in mice that produced different heavy chain repertoires (and corresponding different human heavy chain variable region repertoires) as a result of the human gene repertoire sectoring.
  • FIG. 15 illustrates the improvement in human VH gene segment usage in transcripts (and thus improvement in inactivation of endogenous mouse VH gene segment usage) as human heavy chain locus DNA is inserted in successive BACs for the S1 chimaeric heavy chain locus (human VDJ gene segments from BAC1 only have been inserted) versus the S2 chimaeric heavy chain locus (human VDJ gene segments from BACs1 & 2 have been inserted) and V6 chimaeric heavy chain locus ((human VDJ gene segments from BACs 1 & 6 have been inserted)).
  • BAC1 inserts 6 human VH and all functional human DH and JH gene segments between the 3′ endogenous JH (mouse JH4) and the mouse C-mu.
  • BAC2 inserts an additional 5 human VH gene segments 5′ to the human DNA inserted from the BAC1, resulting in 11 human VH and all functional human DH and JH between the 3′ endogenous JH4 and the mouse C-mu.
  • BAC6 adds 8 human VH gene segments 5′ to the human DNA inserted from BAC1, resulting in 14 human VH and all functional human DH and JH between the 3′ endogenous JH4 and the mouse C-mu. With each insertion, the endogenous mouse heavy chain VDJ region is pushed further upstream (5′) and inactivation is enhanced.
  • BAC6 human gene segments VH3-66, 3-64, 4-61, 4-59, 1-58, 3-53, 5-51 and 3-49.
  • FIG. 16 compares the effect of sectoring a 19 human VH gene segment repertoire across two different mice-types (corresponding to insertion of human gene segment DNA from BACs1+2 (S2 genotype) and BACs1+6 (V6 genotype). Illustrated is the distribution of human VH usage at the transcript level from the mice. Different VH usage was seen resulting in mice that produced different heavy chain repertoires (and corresponding different human heavy chain variable region repertoires) as a result of the human gene repertoire sectoring.
  • FIG. 17 shows the human gene segment repertoires contained in the first, second, third and fourth BACs used to construct four different mice lines, K1, K2, K3 and K4:—
  • K4 comprises an endogenous Ig kappa locus in which has been inserted the human gene segments V ⁇ 3D-40 to 3D-7, V ⁇ 2D-40, V ⁇ 1D-39 and V ⁇ 1-33 to J ⁇ 5.
  • FIG. 18 shows total human V ⁇ gene segment usage versus mouse V ⁇ usage in transcripts from K4 mice.
  • FIG. 19 illustrates the improvement in human V ⁇ gene segment usage in transcripts (and thus improvement in inactivation of endogenous mouse V ⁇ gene segment usage) as human kappa locus DNA is inserted in successive BACs 1-4.
  • FIG. 20 compares the effect of sectoring a 6, 13, 19 and 29 human V ⁇ gene segment repertoire across four different mice-types (corresponding to insertion of human gene segment DNA from BAC1 only (K1 genotype), BACs1+2 (K2 genotype), BACs1+2+3 (K3 genotype) and BACs1+2+3+4 (K4 genotype). Illustrated is the distribution of human V ⁇ usage at the transcript level from the mice. Different V ⁇ usage was seen resulting in mice that produced different kappa chain repertoires (and corresponding different human kappa variable region repertoires) as a result of the human gene repertoire sectoring.
  • V ⁇ variants mentioned are known in the art (and incorporated herein by reference for possible inclusion in claims herein), eg, known from the IMGT database mentioned herein or 1000 Genomes database (release 1, version 3, 16 Mar. 2012), or are disclosed in the Sequence Listing herein (for variants labelled *d01).
  • NB V ⁇ 3-20 and V ⁇ 3D-15 were not present in the BACs or mice.
  • FIG. 21 compares the effect of sectoring the human J ⁇ gene segment repertoire across four different mice-types (K1, K2, K3 and K4). Illustrated is the distribution of human J ⁇ usage at the transcript level from the mice. Different J ⁇ usage was seen resulting in mice that produced different kappa chain repertoires (and corresponding different human kappa variable region repertoires) as a result of the human gene repertoire sectoring.
  • the present invention allows one to dedicate the B-cell compartment of individual vertebrates within the population to a gene segment sub-repertoire from one sector of the overall desired repertoire to be accessed.
  • the invention in one embodiment enables the skilled person to restrict individual vertebrates in the population to one type of light chain (lambda or kappa) predominantly or exclusively, thereby biasing the antibody repertoire within that individual vertebrate to (predominantly) lambda- or kappa-type antibodies.
  • the finite capacity of the B-cell compartment (the accessible compartment being up to around 2 ⁇ 10 8 B-cells in a mouse) is controlled artificially to produce antibodies of substantially only the lambda or kappa type in this example.
  • a population of such vertebrates ie, a combination of lambda-biased vertebrates with kappa-biased vertebrates
  • This repertoire comprises a sub-repertoire of lambda-type antibodies from vertebrates biased to lambda chain expression as well as a second sub-repertoire of kappa-type antibodies from vertebrates biased to kappa chain expression.
  • mice diversity is limited by the size of the accessible B-cell compartment so that it is not possible to sample more of the potential VJ light chain repertoire (which is greater than the approximately 2 ⁇ 10 8 accessible B-cells available in a mouse to express antibodies).
  • VJ light chain repertoire which is greater than the approximately 2 ⁇ 10 8 accessible B-cells available in a mouse to express antibodies.
  • these prior art mice are used in methods that sample only a very small amount of the large potential diversity encoded by recombination of the lambda and kappa V and J gene segments. See the examples below for an illustration.
  • a gene segment eg, a V or J gene segment
  • a gene segment upstream of a constant region in an Ig locus in any configuration of the invention enables the gene segment to be recombined and expressed in an immunoglobulin chain comprising sequence encoded by the constant region of the locus.
  • sub-repertoires can comprise common human gene segments from the overall repertoire (ie, overlapping sub-repertoires) or comprise no common gene segments from the repertoire (ie, non-overlapping sub-repertoires).
  • the antibody heavy chain sequence repertoire of the invention is a repertoire of RNA sequences (eg, mRNA) each comprising a sequence derived from the recombination of a human VH gene segment with a D and JH gene segment and a constant region (eg, a C-mu gene segment or C-gamma gene segment).
  • the antibody heavy chain sequence repertoire is a repertoire of RNA sequences (eg, mRNA) each encoding an antibody heavy chain.
  • the antibody heavy chain sequence repertoire is a repertoire of antibody heavy chains (eg, provided as part of antibodies).
  • an antibody light chain sequence repertoire can be a repertoire of RNA sequences (eg, mRNA) each comprising a sequence derived from the recombination of a human VL gene segment (eg, V ⁇ or V ⁇ ) with a JiL gene segment (eg, J ⁇ or J ⁇ respectively) and a constant region (eg, a C-kappa gene segment or C-lambda gene segment).
  • the antibody light chain sequence repertoire is a repertoire of RNA sequences (eg, mRNA) each encoding an antibody light chain.
  • the antibody light chain sequence repertoire is a repertoire of antibody light chains (eg, provided as part of antibodies).
  • antibody includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., dAb, Fab, F(ab′)2, and Fv).
  • antibody also includes H2 antibodies that comprise a dimer of a heavy chain (5′-VH-(optional Hinge)-CH2-CH3-3′) and are devoid of a light chain (akin to naturally-occurring H2 antibodies; see, eg, Nature. 1993 Jun.
  • RNA produced from the transgenic heavy chain locus encodes for heavy chains that re devoid of a CH1 gene segment and comprise no functional antibody light chain.
  • RNA produced from the transgenic heavy chain locus encodes for VH single variable domains (dAbs; domain antibodies). These can optionally comprise a constant region.
  • the heavy chain sequence repertoire encodes for heavy chains that re devoid of a CH1 gene segment and comprise no functional antibody light chain.
  • the heavy chain sequence repertoire encodes a repertoire of VH single variable domains (dAbs; domain antibodies). These can optionally comprise a constant region.
  • a repertoire comprises a plurality of different members (thus, for example, a heavy chain sequence repertoire comprises a plurality of different heavy chain sequences, such as sequences differing in their variable regions).
  • a repertoire comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 members.
  • a repertoire of antibody chain sequences or antibodies comprises or consists of at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 antibody chain sequences or antibodies respectively.
  • a repertoire comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 different members.
  • a repertoire of gene segments (eg, human VH gene segments or VL gene segments or V ⁇ gene segments or V ⁇ gene segments) comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50 gene segments.
  • all of the gene segments are different from each other.
  • a repertoire of human gene segments used in the invention comprises additionally non-human gene segments, eg, non-human vertebrate gene segments and/or synthetic gene segments. Such combinations of gene segments is desirable to enhance possible variable region diversity.
  • a population comprises a plurality of different members.
  • a population of transgenic non-human vertebrates eg, mice or rats
  • the genomes can, for example, differ in their respective repertoire of human gene segments (eg, human VH gene segments).
  • a population of non-human vertebrates comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 10 3 non-human vertebrates.
  • the genomes of vertebrates are different, eg, the genomes comprise different repertoires of human heavy chain and/or light chain locus gene segments, eg, VH or VL gene segments; or eg, a first of the genomes comprises human lambda locus gene segments (eg, a repertoire of human V ⁇ and/or J ⁇ ) and (substantially) no human kappa locus gene segments and a second of the genomes comprises human kappa locus gene segments (eg, a repertoire of human V ⁇ and/or J ⁇ ) and optionally (substantially) no human lambda locus gene segments).
  • a first of the genomes comprises human lambda locus gene segments (eg, a repertoire of human V ⁇ and/or J ⁇ ) and (substantially) no human kappa locus gene segments
  • a second of the genomes comprises human kappa locus gene segments (eg, a repertoire of human V ⁇ and/or J ⁇ ) and optionally (substantially) no human lambda
  • each vertebrate is a non-human mammal.
  • the vertebrate is a mouse, rat, rabbit, Camelid (eg, a llama, alpaca or camel), chicken or shark.
  • all vertebrates are of the same vertebrate species, eg, all mice or all rats.
  • the inserted human genes may be derived from the same individual or different individuals, or be synthetic or represent human consensus sequences.
  • human heavy chain gene segments are inserted into the genome so that they are placed under control of the host regulatory sequences (eg, enhancers, promoters and/or switches) or other (non-human, non-host) sequences.
  • host regulatory sequences eg, enhancers, promoters and/or switches
  • other (non-human, non-host) sequences e.g., promoters, promoters and/or switches
  • reference to human coding regions includes both human introns and exons, or in another aspect simply exons and no introns, which may be in the form of cDNA.
  • recombineering in ES cells, or other recombinant DNA technologies, to insert a non human-vertebrate (e.g. mouse) promoter or other control region, such as a promoter for a V region, into a BAC containing a human Ig region.
  • a non human-vertebrate e.g. mouse
  • the recombineering step then places a portion of human DNA under control of the mouse promoter or other control region.
  • a (or each) non-human vertebrate of any configuration of the invention is able to generate a diversity of at least 1 ⁇ 10 6 different functional chimaeric immunoglobulin sequence combinations.
  • each constant region is endogenous to the vertebrate and optionally comprises an endogenous switch (when the constant region is a heavy chain constant region).
  • the constant region comprises a Cgamma (CY) gene segment (eg, a human Cgamma) and/or a Smu (Su) switch.
  • CY Cgamma
  • Smu Smu switch.
  • Switch sequences are known in the art, for example, see Nikaido et al, Nature 292: 845-848 (1981) and also WO2011004192, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364, WO2009/076464 and U.S. Pat. No.
  • the constant region comprises an endogenous S gamma switch and/or an endogenous Smu switch.
  • One or more endogenous switch regions can be provided, in one embodiment, by constructing a transgenic immunoglobulin locus in the vertebrate or cell genome in which at least one human V region, at least one human J region, and optionally at least one human D region, or a rearranged VDJ or VJ region, are inserted into the genome in operable connection with a constant region that is endogenous to the vertebrate or cell.
  • the human V(D)J regions or rearranged VDJ or VJ can be inserted in a cis orientation onto the same chromosome as the endogenous constant region.
  • a trans orientation is also possible, in which the human V(D)J regions or rearranged VDJ or VJ are inserted into one chromosome of a pair (eg, the chromosome 6 pair in a mouse or the chromosome 4 in a rat) and the endogenous constant region is on the other chromosome of the pair, such that trans-switching takes place in which the human V(D)J regions or rearranged VDJ or VJ are spliced inoperable linkage to the endogenous constant region.
  • the vertebrate can express antibodies having a chain that comprises a variable region encoded all or in part by human V(D)J or a rearranged VDJ or VJ, together with a constant region (eg, a Cgamma or Cmu) that is endogenous to the vertebrate.
  • a constant region eg, a Cgamma or Cmu
  • Human variable regions are suitably inserted upstream of a non-human vertebrate constant region, the latter comprising all of the DNA required to encode the full constant region or a sufficient portion of the constant region to allow the formation of an effective chimaeric antibody capable of specifically recognising an antigen.
  • the chimaeric antibodies or antibody chains isolated from vertebrates according to the invention have a part of a host constant region sufficient to provide one or more effector functions seen in antibodies occurring naturally in a host vertebrate, for example that they are able interact with Fc receptors, and/or bind to complement.
  • references to a chimaeric antibody or antibody chain having a non-human vertebrate constant region herein therefore is not limited to the complete constant region but also includes chimaeric antibodies or chains which have all of the host constant region, or a part thereof sufficient to provide one or more effector functions.
  • This also applies to non-human vertebrates used in the invention in which human variable region DNA may be inserted into the host genome such that it forms a chimaeric antibody chain with all or part of a host constant region.
  • the whole of a host non-human vertebrate constant region is operably linked to human variable region DNA.
  • the host non-human vertebrate constant region herein is optionally the endogenous host wild-type constant region located at the wild type locus, as appropriate for the heavy or light chain.
  • the human heavy chain DNA is suitably inserted on mouse chromosome 12, suitably adjacent the mouse heavy chain constant region, where the vertebrate is a mouse.
  • the insertion of human DNA is targeted to the region between the J4 exon and the C ⁇ locus in the mouse genome IgH locus, and in one aspect is inserted between coordinates 114,667,090 and 114,665,190, suitably at coordinate 114,667,091, after 114,667,090.
  • the insertion of a human DNA is targeted into mouse chromosome 6 between coordinates 70,673,899 and 70,675,515, suitably at position 70,674,734, or an equivalent position in the lambda mouse locus on chromosome 16.
  • NCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEMBL Release 55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified.
  • Human nucleotides coordinates are those corresponding to GRCh37 (e.g.
  • ENSEMBL Release 55.or are those corresponding to NCBI36, Ensemble release 54 unless otherwise specified.
  • Rat nucleotides are those corresponding to RGSC 3.4 Dec. 2004 ENSEMBL release 55.34w, or Baylor College of Medicine HGSC v3.4 Nov. 2004 (e.g., UCSC rn4, see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified.
  • the host non-human vertebrate constant region for forming the chimaeric antibody may be at a different (non endogenous) chromosomal locus.
  • the inserted human DNA such as the human variable VDJ or VJ region(s) may then be inserted into the non-human genome at a site which is distinct from that of the naturally occurring heavy or light constant region.
  • the native constant region may be inserted into the genome, or duplicated within the genome, at a different chromosomal locus to the native position, such that it is in a functional arrangement with the human variable region such that chimaeric antibodies of the invention can still be produced.
  • the human DNA is inserted at the endogenous host wild-type constant region located at the wild type locus between the host constant region and the host VDJ region.
  • variable region or a gene segment upstream of a constant region means that there is a suitable relative location of the two antibody portions, variable/gene segment and constant, to allow the portions to form a chimaeric antibody or antibody chain in vivo in the vertebrate.
  • the inserted human DNA and host constant region are in operable connection with one another for antibody or antibody chain production.
  • the inserted human DNA is capable of being expressed with different host constant regions through isotype switching.
  • isotype switching does not require or involve trans switching. Insertion of the human variable region DNA on the same chromosome as the relevant host constant region means that there is no need for trans-switching to produce isotype switching.
  • non-human vertebrate constant regions are maintained and in an example at least one non-human vertebrate enhancer or other control sequence, such as a switch region, is maintained in functional arrangement with the non-human vertebrate constant region, such that the effect of the enhancer or other control sequence, as seen in the host vertebrate, is exerted in whole or in part in the transgenic animal.
  • This approach is designed to allow the full diversity of the human locus to be sampled, to allow the same high expression levels that would be achieved by non-human vertebrate control sequences such as enhancers, and is such that signalling in the B-cell, for example isotype switching using switch recombination sites, would still use non-human vertebrate sequences.
  • a non-human vertebrate having such a genome would produce chimaeric antibodies with human variable and non-human vertebrate constant regions, but these are readily humanized, for example in a cloning step.
  • V and/or other gene segments which are naturally inverted in a human genome or are pseudogenes may be omitted from the repertoire provided by the population of the invention.
  • V ⁇ gene segments is the group V ⁇ 2-18, V ⁇ 3-16, V ⁇ 2-14, V ⁇ 3-12, V ⁇ 2-11, V ⁇ 3-10, V ⁇ 3-9, V ⁇ 2-8, V ⁇ 4-3 and V ⁇ 3-1.
  • An example of functional human J ⁇ gene segments is the group J ⁇ 1, J ⁇ 2 and J ⁇ 3; or J ⁇ 1, J ⁇ 2 and J ⁇ 7; or J ⁇ 2, J ⁇ 3 and J ⁇ 7; or J ⁇ 1, J ⁇ 2, J ⁇ 3 and J ⁇ 7.
  • An example of functional human C ⁇ gene segments is the group C ⁇ 1, C ⁇ 2 and C ⁇ 3; or C ⁇ 1, C ⁇ 2 and C ⁇ 7; or C ⁇ 2, C ⁇ 3 and C ⁇ 7; or C ⁇ 1, C ⁇ 2, C ⁇ 3 and C ⁇ 7.
  • germline configuration refers to a germline genomic configuration.
  • human immunoglobulin gene segments of a transgenic immunoglobulin locus are in a germline configuration when the relative order of the gene segments is the same as the order of corresponding gene segments in a human germline genome.
  • the transgenic locus is a heavy chain locus of the invention comprising hypothetical human immunoglobulin gene segments A, B and C, these would be provided in this order (5′ to 3′ in the locus) when the corresponding gene segments of a human germline genome comprises the arrangement 5′-A-B-C-3′.
  • the human immunoglobulin locus elements are in germline configuration when the relative order of the gene segments is the same as the order of corresponding gene segments in a human germline genome and human sequences between the elements are included, these corresponding to such sequences between corresponding elements in the human germline genome.
  • the transgenic locus comprises human elements in the arrangement 5′-A-S1-B-S2-C-S3-3′, wherein A, B and C are human immunoglobulin gene segments and S1-S3 are human inter-gene segment sequences, wherein the corresponding arrangement 5′-A-S1-B-S2-C-S3-3′ is present in a human germline genome.
  • this can be achieved by providing in a transgenic immunoglobulin locus of the invention a DNA insert corresponding to the DNA sequence from A to C in a human germline genome (or the insert comprising the DNA sequence from A to C).
  • the arrangements in human germline genomes and immunoglobulin loci are known in the art (eg, see the IMGT, Kabat and other antibody resources).
  • the Kabat Database (G. Johnson and T. T. Wu, 2002; World Wide Web (www) kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in 1966, the Kabat database publishes aligned sequences of antibodies, T-cell receptors, major histocompatibility complex (MHC) class I and II molecules, and other proteins of immunological interest. A searchable interface is provided by the SeqhuntII tool, and a range of utilities is available for sequence alignment, sequence subgroup classification, and the generation of variability plots. See also Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K., and Foeller, C.
  • MHC major histocompatibility complex
  • IMGT the International ImMunoGeneTics Information System®; M.-P. Lefranc, 2002; World Wide Web (www) imgt.cines.fr).
  • IMGT is an integrated information system that specializes in antibodies, T cell receptors, and MHC molecules of all vertebrate species. It provides a common portal to standardized data that include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, and two-dimensional (2D) and three-dimensional (3D) structures.
  • IMGT includes three sequence databases (IMGT/LIGM-DB, IMGT/MHC-DB, IMGT/PRIMERDB), one genome database (IMGT/GENE-DB), one 3D structure database (IMGT/3Dstructure-DB), and a range of web resources (“IMGT Marie-Paule page”) and interactive tools.
  • V-BASE (I. M. Tomlinson, 2002; World Wide Web (www) mrc-cpe.cam.ac.uk/vbase).
  • V-BASE is a comprehensive directory of all human antibody germline variable region sequences compiled from more than one thousand published sequences. It includes a version of the alignment software DNAPLOT (developed by Hans-Helmar Althaus and Werner Müller) that allows the assignment of rearranged antibody V genes to their closest germline gene segments.
  • Antibodies Structure and Sequence (A. C. R. Martin, 2002; World Wide Web (www) bioinf.org.uk/abs). This page summarizes useful information on antibody structure and sequence. It provides a query interface to the Kabat antibody sequence data, general information on antibodies, crystal structures, and links to other antibody-related information. It also distributes an automated summary of all antibody structures deposited in the Protein Databank (PDB). Of particular interest is a thorough description and comparison of the various numbering schemes for antibody variable regions.
  • PDB Protein Databank
  • AAAAA A Ho's Amazing Atlas of Antibody Anatomy; A. Honegger, 2001; World Wide Web (www) unizh.ch/ ⁇ antibody.
  • This resource includes tools for structural analysis, modelling, and engineering. It adopts a unifying scheme for comprehensive structural alignment of antibody and T-cell-receptor sequences, and includes Excel macros for antibody analysis and graphical representation.
  • WAM Web Antibody Modeling; N. Whitelegg and A. R. Rees, 2001; World Wide Web (www) antibody.bath.ac.uk). Hosted by the Centre for Protein Analysis and Design at the University of Bath, United Kingdom. Based on the AbM package (formerly marketed by Oxford Molecular) to construct 3D models of antibody Fv sequences using a combination of established theoretical methods, this site also includes the latest antibody structural information.
  • the Antibody Resource Page (The Antibody Resource Page, 2000; World Wide Web (www) antibodyresource.com). This site describes itself as the “complete guide to antibody research and suppliers.” Links to amino acid sequencing tools, nucleotide antibody sequencing tools, and hybridoma/cell-culture databases are provided.
  • Humanization bY Design J. Saldanha, 2000; World Wide Web (www) people.cryst.bbk.ac.uk/ ⁇ ubcg07s.
  • This resource provides an overview on antibody humanization technology.
  • the most useful feature is a searchable database (by sequence and text) of more than 40 published humanized antibodies including information on design issues, framework choice, framework back-mutations, and binding affinity of the humanized constructs.
  • Samples from which B-cells can be obtained include but are not limited to blood, serum, spleen, splenic tissue, bone marrow, lymph, lymph node, thymus, and appendix.
  • Antibodies and immunoglobulin chains can be obtained from each of the previous-mentioned samples and also from the following non-limiting list of B-cells, ascites fluid, hybridomas, and cell cultures.
  • a human variable region of a heavy chain can be derived from recombination of human VH, D and JH gene segments and this reflects the in vivo recombination of these gene segments in, for example, a transgenic heavy chain locus according to the invention with any accompanying mutation (eg, junctional mutation).
  • the genome of a or each vertebrate has been modified to prevent or reduce the expression of fully-endogenous antibody.
  • suitable techniques for doing this can be found in WO2011004192, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364, WO2009/076464, EP1399559 and U.S. Pat. No. 6,586,251, the disclosures of which are incorporated herein by reference.
  • the non-human vertebrate VDJ region of the endogenous heavy chain immunoglobulin locus, and optionally VJ region of the endogenous light chain immunoglobulin loci have been inactivated.
  • all or part of the non-human vertebrate VDJ region is inactivated by inversion in the endogenous heavy chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus.
  • all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous kappa chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus.
  • all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous lambda chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus.
  • the endogenous heavy chain locus is inactivated in this way as is one or both of the endogenous kappa and lambda loci.
  • the or each vertebrate has been generated in a genetic background which prevents the production of mature host B and T lymphocytes, optionally a RAG-1-deficient and/or RAG-2 deficient background. See U.S. Pat. No. 5,859,301 for techniques of generating RAG-1 deficient animals.
  • the immunoglobulin loci of the vertebrates differ only in the repertoire of said human gene segments.
  • immunoglobulin (Ig) is used interchangeably with “antibody” herein.
  • an “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly).
  • the isolated polypeptide is free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvable or approved standard.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
  • antibody fragment comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include dAb, Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • an antibody that “specifically binds to” or is “specific for” a particular polypeptide, antigen, or epitope is one that binds to that particular polypeptide, antigen, or epitope without substantially binding to other polypeptides, antigens or epitopes.
  • binding to the antigen or epitope is specific when the antibody binds with a K D of 100 ⁇ M or less, 10 ⁇ M or less, 1 ⁇ M or less, 100 nM or less, eg, 10 nM or less, 1 nM or less, 500 pM or less, 100 pM or less, or 10 pM or less.
  • the binding affinity (K D ) can be determined using standard procedures as will be known by the skilled person, eg, binding in ELISA and/or affinity determination using surface plasmon resonance (eg, BiacoreTM or KinExATM solution phase affinity measurement which can detect down to fM affinities (Sapidyne Instruments, Idaho)).
  • surface plasmon resonance eg, BiacoreTM or KinExATM solution phase affinity measurement which can detect down to fM affinities (Sapidyne Instruments, Idaho)
  • “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • a “pharmaceutically acceptable carrier, excipient, or adjuvant” refers to an carrier, excipient, or adjuvant that can be administered to a subject, together with an agent, e.g., any antibody or antibody chain described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • the constant region comprises a mouse or rat S ⁇ switch and optionally a mouse Cu region.
  • the constant region is provided by the constant region endogenous to the mouse, eg, by inserting human V(D)J region sequences into operable linkage with the endogenous constant region of a mouse genome or mouse cell genome.
  • the constant region comprises a mouse or rat Su switch and optionally a rat Cu region.
  • the constant region is provided by the constant region endogenous to the rat, eg, by inserting human V(D)J region sequences into operable linkage with the endogenous constant region of a rat genome or rat cell genome.
  • the genome comprises an antibody light chain transgene which comprises all or part of the human Ig ⁇ locus including at least one human J ⁇ region and at least one human C ⁇ region, optionally C ⁇ 6 and/or C ⁇ 7.
  • the transgene comprises a plurality of human J ⁇ regions, optionally two or more of J ⁇ 1, J ⁇ 2, J ⁇ 6 and J ⁇ 7, optionally all of J ⁇ 1, J ⁇ 2, J ⁇ 6 and J ⁇ 7.
  • the human lambda immunoglobulin locus comprises a unique gene architecture composed of serial J-C clusters.
  • the invention in optional aspects employs one or more such human J-C clusters inoperable linkage with the constant region in the transgene, eg, where the constant region is endogenous to the non-human vertebrate or non-human vertebrate cell.
  • the transgene comprises at least one human J ⁇ -C ⁇ cluster, optionally at least J ⁇ 7-C ⁇ 7.
  • the construction of such transgenes is facilitated by being able to use all or part of the human lambda locus such that the transgene comprises one or more J-C clusters in germline configuration, advantageously also including intervening sequences between clusters and/or between adjacent J and C regions in the human locus. This preserves any regulatory elements within the intervening sequences which may be involved in VJ and/or JC recombination and which may be recognised by AID (activation-induced deaminase) or AID homologues.
  • endogenous regulatory elements are involved in CSR (class-switch recombination) in the non-human vertebrate
  • these can be preserved by including in the transgene a constant region that is endogenous to the non-human vertebrate.
  • Such design elements are advantageous for maximising the enzymatic spectrum for SHM (somatic hypermutation) and/or CSR and thus for maximising the potential for antibody diversity.
  • the lambda transgene comprises a human EX enhancer.
  • the kappa transgene comprises a human EK enhancer.
  • the heavy chain transgene comprises a heavy chain human enhancer.
  • the constant region is endogenous to the non-human vertebrate or derived from such a constant region.
  • the vertebrate is a mouse and the constant region is endogenous to the mouse.
  • the vertebrate is a rat and the constant region is endogenous to the rat.
  • the heavy chain transgene comprises a plurality human IgH V regions, a plurality of human D regions and a plurality of human J regions.
  • the vertebrate comprises a heavy chain further transgene, the further transgene comprising at least one human IgH V region, at least one human D region and at least one human J region.
  • An aspect provides a method of isolating an antibody or nucleotide sequence encoding said antibody, the method comprising
  • Such joining can be effected by techniques readily available in the art, such as using conventional recombinant DNA and RNA technology as will be apparent to the skilled person. See e.g. Sambrook, J and Russell, D. (2001, 3′d edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).
  • an immunogenic amount of the antigen is delivered.
  • the invention also relates to a method for detecting a target antigen comprising detecting an antibody produced as above with a secondary detection agent which recognises a portion of that antibody.
  • Isolation of the antibody in step (b) can be carried out using conventional antibody selection techniques, eg, panning for antibodies against antigen that has been immobilised on a solid support, optionally with iterative rounds at increasing stringency, as will be readily apparent to the skilled person.
  • step (b) the amino acid sequence of the heavy and/or the light chain variable regions of the antibody are mutated to improve affinity for binding to said antigen. Mutation can be generated by conventional techniques as will be readily apparent to the skilled person, eg, by error-prone PCR. Affinity can be determined by conventional techniques as will be readily apparent to the skilled person, eg, by surface plasmon resonance, eg, using BiacoreTM.
  • step (b) after step (b) the amino acid sequence of the heavy and/or the light chain variable regions of the antibody are mutated to improve one or more biophysical characteristics of the antibody, eg, one or more of melting temperature, solution state (monomer or dimer), stability and expression (eg, in CHO or E. coli ).
  • An aspect provides an antibody produced by the method of the invention, optionally for use in medicine, eg, for treating and/or preventing a medical condition or disease in a patient, eg, a human.
  • nucleotide sequence encoding the antibody of the invention, optionally wherein the nucleotide sequence is part of a vector.
  • Suitable vectors will be readily apparent to the skilled person, eg, a conventional antibody expression vector comprising the nucleotide sequence together in operable linkage with one or more expression control elements.
  • An aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the invention and a diluent, excipient or carrier, optionally wherein the composition is contained in an IV container (eg, and IV bag) or a container connected to an IV syringe.
  • An aspect provides the use of the antibody of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition in a patient, eg a human.
  • the invention relates to a method for producing an antibody specific to a desired antigen the method comprising immunizing a population of non-human vertebrates as above with a predetermined antigen and recovering a chimaeric antibody (see e.g. Harlow, E. & Lane, D. 1998, 5 th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259).
  • an immunogenic amount of the antigen is delivered.
  • the invention also relates to a method for detecting a target antigen comprising detecting an antibody produced as above with a secondary detection agent which recognises a portion of that antibody.
  • the invention in a further aspect relates to a method for producing a fully humanised antibody comprising immunizing a population of non-human vertebrates as above with a predetermined antigen, recovering a chimaeric antibody or cells expressing the antibody, and then replacing the non-human vertebrate constant region with a human constant region.
  • This can be done by standard cloning techniques at the DNA level to replace the non-human vertebrate constant region with an appropriate human constant region DNA sequence—see e.g. Sambrook, J and Russell, D. (2001, 3′d edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).
  • the invention relates to humanised antibodies and antibody chains produced according to the present invention, both in chimaeric and fully humanised form, and use of said antibodies in medicine.
  • the invention also relates to a pharmaceutical composition comprising such an antibody and a pharmaceutically acceptable carrier or other excipient.
  • Antibody chains containing human sequences such as chimaeric human-non-human antibody chains, are considered humanised herein by virtue of the presence of the human protein coding regions region.
  • Fully humanised antibodies may be produced starting from DNA encoding a chimaeric antibody chain of the invention using standard techniques.
  • chimaeric antibodies or antibody chains generated using the present invention may be manipulated, suitably at the DNA level, to generate molecules with antibody-like properties or structure, such as a human variable region from a heavy chain absent a constant region, for example a domain antibody; or a human variable region with any constant region from either heavy or light chain from the same or different species; or a human variable region with a non-naturally occurring constant region; or human variable region together with any other fusion partner.
  • the invention relates to all such chimaeric antibody derivatives derived from chimaeric antibodies identified using the present invention.
  • the invention relates to use of a population of non-human vertebrates of the present invention in the analysis of the likely effects of drugs and vaccines in the context of a quasi-human antibody repertoire.
  • the invention also relates to a method for identification or validation of a drug or vaccine, the method comprising delivering the vaccine or drug to a population of vertebrates of the invention and monitoring one or more of: the immune response, the safety profile; the effect on disease.
  • the invention also relates to a kit comprising an antibody or antibody derivative as disclosed herein and either instructions for use of such antibody or a suitable laboratory reagent, such as a buffer, antibody detection reagent.
  • a suitable laboratory reagent such as a buffer, antibody detection reagent.
  • the invention also relates to a method for making an antibody, or part thereof, the method comprising providing:
  • nucleic acid encoding an antibody, or a part thereof, obtained using the population of the present invention (i) a nucleic acid encoding an antibody, or a part thereof, obtained using the population of the present invention; or (ii) sequence information from which a nucleic acid encoding an antibody obtained using the population of the present invention, or part thereof, can be expressed to allow an antibody to be produced.
  • each vertebrate of the population of the first aspect of the invention is a non-human vertebrate, mouse or rat, whose genome comprises
  • transgenic heavy chain locus (a) said transgenic heavy chain locus; and (b) an antibody kappa light chain locus transgene and/or an antibody lambda chain locus transgene; wherein all of the V, D and J in said transgenes are human V, D and J; wherein endogenous antibody heavy and light chain expression has been inactivated; and optionally wherein said genome is homozygous for said transgenic heavy and light chain loci.
  • the kappa and lambda chain transgenic loci comprise constant regions of said non-human vertebrate species capable of pairing with the constant region of the heavy chain.
  • the kappa chain transgenic loci comprises a substantially complete human functional V ⁇ and J ⁇ repertoire; and the lambda chain transgene comprises a substantially complete human functional V ⁇ and J ⁇ repertoire.
  • the term “endogenous” in relation to a non-human vertebrate indicates that the constant region etc is a type of constant region etc that is normally found in the vertebrate (as opposed to an exogenous constant region whose sequence is not normally found in such a vertebrate, eg a human sequence).
  • the endogenous constant region can be those encoded by the wild-type genome of the non-human vertebrate. So, in an example wherein the vertebrate is a mouse, the endogenous constant region would be a mouse constant region. Going further, the endogenous regions are, in an example, strain-matched to the vertebrate.
  • the vertebrate cell is a mouse 129 ES cell, the endogenous constant region would be mouse 129 constant region.
  • the vertebrate is a JM8 strain mouse, the endogenous constant region would be mouse JM8 constant region.
  • the vertebrate is a Black 6 mouse, the endogenous constant region would be mouse Black 6 constant region.
  • the constant region of the heavy chain transgenic locus is a non-human vertebrate constant region (eg, mouse or rat constant region).
  • the constant region is endogenous to said non-human vertebrate.
  • the constant region of the heavy chain transgene is human constant region.
  • the constant region is human and devoid of a CH1. This is useful for producing human H2 antibodies (especially when the vertebrate is not capable of expressing light chains).
  • the constant region is or comprises a Cmu, eg, a mouse or rat Cmu.
  • the Cmu is an endogenous mouse Cmu.
  • the transgenic heavy chain locus in an example, comprises a Smu switch 5′ of the Cmu and a Cgamma 3′ of the Cmu, with a S gamma switch between the Cmu and Cgamma.
  • the Cmu, Cgamma and switches are endogenous mouse C regions and switches.
  • the C regions and switches are mouse 129 C regions and switches; or the C regions and switches are mouse Black 6 C regions and switches.
  • the S gamma and C regions are mouse S gamma and C regions, and the Smu is a rat Smu.
  • each vertebrate of the population is a mouse whose genetic background is selected from mouse strains C57BL/6, M129 such as 129/SV, BALB/c, and any hybrid of C57BL/6, M129 such as 129/SV, or BALB/c.
  • each of these vertebrates have the same genetic background but two or more of the vertebrates of the population differ in their human gene segment repertoires as per the invention.
  • the invention relates to a method of providing a synthetic antibody heavy chain sequence repertoire in a population of non-human vertebrates, the method comprising
  • the population provides an overall heavy chain repertoire comprising the heavy chain sequence repertoires of the first and second vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more heavy chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • VH gene segments of a repertoire can, in one embodiment, be recombined VH, ie, provided as part of a variable region sequence derived from the recombination of human VH with D and JH (eg, where the VH, D and JH are human).
  • the population comprises a third non-human vertebrate, the third vertebrate comprising a transgenic heavy chain locus comprising one or more human VH gene segments (third VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; wherein the third VH gene sub-repertoire is different from the first and second VH gene sub-repertoires, whereby the third vertebrate can produce a heavy chain sequence repertoire that is different from the heavy chain sequence repertoire produced by the first and second vertebrates.
  • the population provides an overall heavy chain repertoire comprising the heavy chain sequence repertoires of the first, second and third vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more heavy chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • VH gene segment repertoire provided by said population comprises a substantially complete repertoire of functional human VH gene segments; optionally providing at least 6 different human JH gene segments, 27 different human D segments and at least 40 different human VH gene segments.
  • the VH gene segment repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human VH gene segments.
  • each transgenic heavy chain locus are human JH gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human JH gene segments.
  • each heavy chain locus comprises at least 2, 3, 4, 5 or 6 different human JH gene segments.
  • each transgenic heavy chain locus are human D gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human D gene segments.
  • each heavy chain locus comprises at least 5, 10, 15, 20, 25, 26 or 27 different human D gene segments.
  • the heavy chain loci of said vertebrates comprise identical human D and JH gene segment repertoires, but differ in their VH gene repertoires.
  • each heavy chain locus comprises at least two human JH gene segments selected from the group consisting of J1, J2, J3, J4, J5 and J6; optionally all of the gene segments of the group.
  • each vertebrate comprises human VH gene segments selected from the group consisting of V6-1, V1-2, V1-3, V4-4, V7-41, V2-5, V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46, V3-48, V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74; wherein the VH gene repertoire comprises a substantially complete human functional VH gene repertoire.
  • endogenous antibody heavy chain expression has been inactivated in the vertebrates.
  • endogenous heavy chains ie, heavy chains whose variable regions are derived from recombination of non-human vertebrate V, D and J gene segments.
  • the method of the invention comprises the step of immunising the vertebrates of the population with the same antigen (eg, a human antigen).
  • the vertebrates are a population and are used as such.
  • immunisation of two, more or all of said vertebrates is separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day.
  • the vertebrates are a population and are used as such.
  • the method of the invention comprises the step of selecting one or more heavy chains or antibodies from each of said immunised vertebrates on the basis of a common desired antibody or heavy chain characteristic (eg, binding affinity for said antigen), wherein the selected antibodies or heavy chains comprise heavy chain variable region sequences derived from the human VH gene segment repertoire provided by the population.
  • a common desired antibody or heavy chain characteristic eg, binding affinity for said antigen
  • the selected antibodies or heavy chains provide a repertoire of selected antibodies or heavy chains (selected repertoire), the method further comprising selecting one or more antibodies or heavy chains from the selected repertoire on the basis of a desired antibody or heavy chain characteristic (eg, binding affinity for said antigen or a different antigen (eg, a related antigen; which is useful for producing bispecific antibodies); or on the basis of the epitope bound by the antibody or heavy chain); optionally wherein the selected repertoire is formed by pooling the selected antibodies or heavy chains.
  • a desired antibody or heavy chain characteristic eg, binding affinity for said antigen or a different antigen (eg, a related antigen; which is useful for producing bispecific antibodies)
  • the selected repertoire is formed by pooling the selected antibodies or heavy chains.
  • the vertebrates share the same genetic background, with the exception of the heavy chain loci thereof (and optionally one or more of the light chain loci thereof).
  • vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA (eg, human heavy chain V, D and J gene segments and/or human light chain V and J gene segments), the ancestor stem cells being identical or related (eg, clonally related); optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • human immunoglobulin locus DNA eg, human heavy chain V, D and J gene segments and/or human light chain V and J gene segments
  • the ancestor stem cells being identical or related (eg, clonally related)
  • the genome of the ancestor stem cells comprise a common sequence junction that is a junction
  • the genomes comprise a common junction within or at the boundary of one or more of their immunoglobulin chain loci (eg, heavy chain loci and/or light chain loci).
  • the vertebrates of the population are mice whose genomes comprise a common human-mouse DNA junction within their heavy chain loci and/or one or more light chain loci. This is indicative that the mice form a population.
  • the vertebrates can all share the same genetic background with the exception of one or more human gene segment repertoires in their genomes.
  • the invention provides a method of providing a synthetic antibody light chain sequence repertoire in a population of non-human vertebrates, the method comprising
  • the population provides an overall light chain repertoire comprising the light chain sequence repertoires of the first and second vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more light chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • VL gene segments of a repertoire can, in one embodiment, be recombined VL, ie, provided as part of a variable region sequence derived from the recombination of human VL with JL (eg, where the VL and JL are human).
  • the population comprises a third non-human vertebrate, the third vertebrate comprising a transgenic light chain locus comprising one or more human VL gene segments (third VL gene sub-repertoire) and J segments operably connected upstream of a constant region; wherein the third VL gene sub-repertoire is different from the first and second VL gene sub-repertoires, whereby the third vertebrate can produce a light chain sequence repertoire that is different from the light chain sequence repertoire produced by the first and second vertebrates.
  • the population provides an overall light chain repertoire comprising the light chain sequence repertoires of the first, second and third vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more light chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • each transgenic light chain locus are human JL gene segments; optionally wherein each light chain locus comprises a substantially complete functional repertoire of human J ⁇ or J ⁇ gene segments (eg, each transgenic locus comprises human V ⁇ gene segments and J ⁇ gene segments, optionally a substantially complete functional repertoire of human J ⁇ gene segments; or each transgenic locus comprises human V ⁇ gene segments and J ⁇ gene segments, optionally a substantially complete functional repertoire of human J ⁇ gene segments).
  • the repertoire provided by said population comprises a substantially complete repertoire of functional human V ⁇ gene segments; optionally providing at least 5 different human J ⁇ gene segments and at least at least 40 different human V ⁇ gene segments.
  • the repertoire if provided by a population of non-human vertebrates of the invention, wherein each vertebrate comprises an IgK locus whose V ⁇ repertoire consists of 30 or less, 20 or less, 14 or less or 6 or less human V ⁇ gene segment types, as shown in the examples.
  • the repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human V ⁇ gene segments.
  • the repertoire provided by said population comprises a substantially complete repertoire of functional human V ⁇ gene segments; optionally providing at least 5 different human J ⁇ gene segments and at least 40 different human V ⁇ gene segments.
  • the repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human V ⁇ gene segments.
  • each light chain locus comprises at least 2, 3, 4, 5 or 6 different human J ⁇ or J ⁇ gene segments.
  • the light chain loci of said vertebrates comprise identical human JL gene segment repertoires, but differ in their VL gene repertoires.
  • endogenous antibody kappa and/or lambda light chain expression has been inactivated in the vertebrates.
  • said transgenic light chain loci of the vertebrates are kappa light chain loci (at the endogenous kappa loci, ie, corresponding to the position of a kappa locus in a wild-type non-human vertebrate genome).
  • a transgenic kappa locus can comprise human V ⁇ gene segments and J ⁇ gene segments upstream of a constant region (eg, a CH, C ⁇ or C ⁇ gene segment; optionally which is an endogenous gene segment).
  • a transgenic kappa locus can comprise human V ⁇ gene segments and J ⁇ gene segments upstream of a constant region (eg, a CH, C ⁇ or C ⁇ gene segment; optionally which is an endogenous gene segment).
  • said transgenic light chain loci of the vertebrates are lambda light chain loci (at the endogenous lambda loci, ie, corresponding to the position of a lambda locus in a wild-type non-human vertebrate genome).
  • a transgenic lambda locus can comprise human V ⁇ gene segments and J ⁇ gene segments upstream of a constant region (eg, a CH, C ⁇ or C ⁇ gene segment; optionally which is an endogenous gene segment).
  • a transgenic lambda locus can comprise human V ⁇ gene segments and J ⁇ gene segments upstream of a constant region (eg, a CH, C ⁇ or C ⁇ gene segment; optionally which is an endogenous gene segment).
  • the method of the invention comprises the step of immunising the vertebrates of the population with the same antigen (eg, a human antigen).
  • the vertebrates are a population and are used as such.
  • immunisation of two, more or all of said vertebrates is separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day.
  • the vertebrates are a population and are used as such.
  • the method of the invention comprises the step of selecting one or more light chains or antibodies from each of said immunised vertebrates on the basis of a common desired antibody or heavy chain characteristic (eg, binding affinity for said antigen), wherein the selected antibodies or light chains comprise light chain variable region sequences derived from the human VL gene segment repertoire provided by the population.
  • a common desired antibody or heavy chain characteristic eg, binding affinity for said antigen
  • the selected antibodies or light chains provide a repertoire of selected antibodies or light chains (selected repertoire), the method further comprising selecting one or more antibodies or light chains from the selected repertoire on the basis of a desired antibody or light chain characteristic (eg, binding affinity for said antigen or a different antigen (eg, a related antigen; which is useful for producing bispecific antibodies); or on the basis of the epitope bound by the antibody or light chain); optionally wherein the selected repertoire is formed by pooling the selected antibodies or light chains.
  • a desired antibody or light chain characteristic eg, binding affinity for said antigen or a different antigen (eg, a related antigen; which is useful for producing bispecific antibodies)
  • the selected repertoire is formed by pooling the selected antibodies or light chains.
  • Pooling refers to providing a combination or collection for further use (eg, for further selection of members of the combination or collection on the basis of desirable antibody or antibody chain characteristic).
  • the members are physically pooled (ie, mixed in a single or a relatively small number of containers, eg, two, three, four, five or six containers).
  • the vertebrates share the same genetic background, with the exception of said transgenic light chain loci thereof (and optionally one or more of the heavy chain loci thereof).
  • the vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related; optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the invention provides a method of selecting an antibody that binds a predetermined antigen (eg, a human antigen), the method comprising
  • a sub-repertoire of antibodies (kappa sub-repertoire) whose light chain variable regions are produced by rearrangement of a human V ⁇ gene segment with a human J L gene segment; (b) selecting one or more antibodies from the lambda sub-repertoire according to a desired antibody characteristic (eg, binding affinity for said antigen); (c) selecting one or more antibodies from the kappa sub-repertoire according to said desired antibody characteristic; wherein a repertoire (second repertoire) of selected lambda and kappa antibodies is produced, the antibodies of the second repertoire comprising human variable regions that bind said antigen; and (d) Selecting one or more antibodies from said second repertoire on the basis of a desired antibody characteristic (eg, binding affinity for said antigen); Wherein in step (a)(i) the lambda sub-repertoire is produced by immunisation of one or more non-human vertebrates (optionally mice or rats) (lambda vertebrates) with said antigen, wherein the lambda verte
  • Each sub-repertoire comprises at least two antibodies, for example, each sub-repertoire comprises or consists of at least 10, 15, 20, 50, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 antibodies.
  • Examples of a desirable antibody characteristic are affinity for binding a predetermined antigen or epitope (eg, as determined by surface plasmon resonance), completion with a known antibody for binding to a predetermined antigen or epitope, epitopic specificity of the antibody (eg, as determined by X-ray crystallography, competition with a known antibody for antigen binding wherein the known antibody specifically binds to the antigen (eg, as determined by surface plasmon resonance, eg, BiacoreTM), performance in ELISA or another immunoassay, a desirable biophysical characteristic (eg, melting temperature, pI, solution state, degree of aggregation, storage profile etc).
  • affinity for binding a predetermined antigen or epitope eg, as determined by surface plasmon resonance
  • completion with a known antibody for binding to a predetermined antigen or epitope eg, epitopic specificity of the antibody (eg, as determined by X-ray crystallography, competition with a known antibody for antigen binding wherein the known antibody specifically
  • the lambda-type antibodies can comprise any constant region, eg, C ⁇ , C ⁇ or CH (optionally wherein the C is endogenous).
  • the kappa-type antibodies can comprise any constant region, eg, C ⁇ , C ⁇ or CH (optionally wherein the C is endogenous).
  • Methods of immunisation for use in the invention are well known to the skilled person and may involve a classic prime-boost regime, RIMMS or any other protocol.
  • An adjuvant may be administered with the antigen, as is known in the art.
  • the second repertoire comprises at least two antibodies, for example, each sub-repertoire comprises or consists of at least 10, 15, 20, 50, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 antibodies.
  • Human heavy chain variable regions are variable regions derived from recombination of a human VH gene segment with a D and JH gene segment (eg, wherein the D and JH gene segments are also human).
  • Human light chain variable regions are variable regions derived from recombination of a human VL gene segment with a JL gene segment (eg, wherein the JL gene segment is also human).
  • the lambda vertebrates express substantially no kappa-type antibodies.
  • the lambda vertebrates express substantially no kappa-type antibodies.
  • less than 10, 5, 4, 3, 2, 1 or 0.5% of kappa antibodies are endogenous.
  • endogenous kappa antibody expression is substantially inactive in the lambda vertebrates.
  • less than 10, 5, 4, 3, 2, 1 or 0.5% of kappa antibodies are endogenous.
  • the kappa vertebrates express substantially no lambda-type antibodies. For example, less than 10, 5, 4, 3, 2, 1 or 0.5% of lambda antibodies are endogenous. Due to the relatively low endogenous lambda expression in mice, when the vertebrates are mice, it may not be necessary to carry out any specific genetic manipulation in the mice to achieve substantially inactive lambda expression.
  • endogenous lambda antibody expression is substantially inactive in the kappa vertebrates.
  • less than 10, 5, 4, 3, 2, 1 or 0.5% of lambda antibodies are endogenous. Due to the relatively low endogenous lambda expression in mice, when the vertebrates are mice, it may not be necessary to carry out any specific genetic manipulation in the mice to achieve substantially inactive lambda expression.
  • formation (eg, first or last immunisation) of the lambda sub-repertoire is separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day from the formation (eg, first or last immunisation) of the kappa sub-repertoire.
  • the vertebrates form a population and are used as such.
  • the second repertoire is formed by pooling the selected lambda and kappa antibodies.
  • lambda and kappa antibodies are selected on the basis of affinity of binding to said antigen (higher affinity being preferable to lower affinity).
  • affinity is determined by surface plasmon resonance.
  • the kappa and lambda vertebrates share the same genetic background, with the exception of the light chain loci thereof (and optionally heavy chain loci thereof).
  • the kappa and lambda vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related; optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the invention provides a population of transgenic non-human vertebrates (optionally mice or rats), wherein the population provides a repertoire of different human VH gene segments, the repertoire being divided between two or more vertebrates of said population,
  • first vertebrate of said population comprising a transgenic heavy chain locus comprising one or more human VH gene segments (first VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; and (b) a second vertebrate of said population comprising a transgenic heavy chain locus comprising one or more human VH gene segments (second VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; (c) wherein the first VH gene sub-repertoire is different from the second VH gene sub-repertoire for expression of first and second antibody heavy chain sequence repertoires respectively that are different from each other, whereby the population provides a synthetic repertoire of antibody heavy chain sequences.
  • the population comprises a third non-human vertebrate, the third vertebrate comprising a transgenic heavy chain locus comprising one or more human VH gene segments (third VH gene sub-repertoire), D segments and J segments operably connected upstream of a constant region; wherein the third VH gene sub-repertoire is different from the first and second VH gene sub-repertoires for expression of a third antibody heavy chain sequence repertoire that is different from the first and second antibody heavy chain sequence repertoires, whereby the population provides a synthetic repertoire of antibody heavy chain sequences.
  • the vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the repertoire provided by said population comprises a substantially complete repertoire of functional human VH gene segments; optionally providing at least 6 different human JH gene segments, 27 different human D segments and at least 40 different human VH gene segments.
  • the human VH gene segment repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human VH gene segments.
  • each transgenic heavy chain locus are human JH gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human JH gene segments.
  • each heavy chain locus comprises at least 2, 3, 4, 5 or 6 different human JH gene segments.
  • each transgenic heavy chain locus are human D gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human D gene segments.
  • each heavy chain locus comprises at least 5, 10, 15, 20, 25, 26 or 27 different human D gene segments.
  • the heavy chain loci of said vertebrates comprise identical human D and JH gene segment repertoires, but differ in their VH gene repertoires.
  • each heavy chain locus comprises at least two human JH gene segments selected from the group consisting of J1, J2, J3, J4, J5 and J6; optionally all of the gene segments of the group.
  • each heavy chain locus comprises at least 10 human D gene segments selected from the group consisting of D1-1, D2-2, D3-3, D4-4, D5-5, D6-6, D1-7, D2-8, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D1-26, D6-25 and D7-27; optionally all of the gene segments of the group.
  • the population comprises a third vertebrate as described above, wherein each vertebrate comprises human VH gene segments selected from the group consisting of V6-1, V1-2, V1-3, V4-4, V7-41, V2-5, V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46, V3-48, V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74; wherein the population provided by the repertoire comprises a substantially complete human functional VH gene repertoire.
  • endogenous antibody heavy chain expression has been inactivated in the vertebrates.
  • two, more or all of said vertebrates of the population have been immunised with the same antigen, wherein two or more of the immunisations are separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day.
  • the vertebrates share the same genetic background, with the exception of the heavy chain loci thereof (and optionally one or more of the light chain loci thereof).
  • the vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related; optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the invention provides a population of transgenic non-human vertebrates (optionally mice or rats), wherein the population provides a repertoire of different human VL gene segments, the repertoire being divided between two or more vertebrates of said population,
  • first vertebrate of said population comprising a transgenic light chain locus comprising one or more human VL gene segments (first VL gene sub-repertoire) and J segments operably connected upstream of a constant region; and a second vertebrate of said population comprising a transgenic light chain locus comprising one or more human VL gene segments (second VL gene sub-repertoire) and J segments operably connected upstream of a constant region; wherein the first VL gene sub-repertoire is different from the second VL gene sub-repertoire for expression of first and second antibody light chain sequence repertoires respectively that are different from each other, whereby the population provides a synthetic repertoire of antibody light chain sequences.
  • the population comprises a third non-human vertebrate, the third vertebrate comprising a transgenic light chain locus comprising one or more human VL gene segments (third VL gene sub-repertoire) and J segments operably connected upstream of a constant region; wherein the third VL gene sub-repertoire is different from the first and second VH gene sub-repertoires for expression of a third antibody light chain sequence repertoire that is different from the first and second antibody light chain sequence repertoires, whereby the population provides a synthetic repertoire of antibody light chain sequences.
  • the third VL gene sub-repertoire is different from the first and second VH gene sub-repertoires for expression of a third antibody light chain sequence repertoire that is different from the first and second antibody light chain sequence repertoires, whereby the population provides a synthetic repertoire of antibody light chain sequences.
  • the vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • each transgenic light chain locus are human JL gene segments; optionally wherein each light chain locus comprises a substantially complete functional repertoire of human J ⁇ or J ⁇ gene segments.
  • the VL gene segment repertoire provided by said population comprises a substantially complete repertoire of functional human V ⁇ gene segments; optionally providing at least 5 different human J ⁇ gene segments and at least 40 different human V ⁇ gene segments.
  • the VL gene segment repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human V ⁇ gene segments.
  • the VL gene segment repertoire provided by said population comprises a substantially complete repertoire of functional human V ⁇ gene segments; optionally providing at least 5 different human J ⁇ gene segments and 30 different human V ⁇ gene segments.
  • the VL gene segment repertoire provided by said population comprises at least 20, 25 or 30 different human V ⁇ gene segments.
  • each light chain locus comprises at least 2, 3, 4, 5 or 6 different human J ⁇ or J ⁇ gene segments.
  • the light chain loci of said vertebrates comprise identical human JL gene segment repertoires, but differ in their VL gene repertoires.
  • endogenous antibody kappa and/or lambda light chain expression has been inactivated in the vertebrates.
  • said transgenic light chain loci of the vertebrates are kappa light chain loci.
  • said transgenic light chain loci of the vertebrates are lambda light chain loci.
  • the light chain loci of said vertebrates comprise identical human JL gene segment repertoires, but differ in their VL gene repertoires.
  • each light chain locus comprises at least 2, 3, 4 or 5 human J ⁇ gene segments or at least 2, 3, 4 or 5 human J ⁇ gene segments.
  • two, more or all of said vertebrates of the population have been immunised with the same antigen, wherein two or more of the immunisations are separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day.
  • the vertebrates share the same genetic background, with the exception of said transgenic light chain loci thereof (and optionally one or more of the heavy chain loci thereof).
  • the vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related; optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the invention provides a population of non-human vertebrates (optionally mice or rats), wherein the genome of each vertebrate comprises:
  • transgenic immunoglobulin heavy chain loci each locus comprising one or more human V gene segments, one or more human D gene segments and one or more human J gene segments upstream of one or more heavy chain constant regions; and
  • transgenic immunoglobulin light chain loci each locus comprising one or more human V L gene segments and one or more human J L gene segments upstream of one or more light chain constant regions;
  • the gene segments in transgenic heavy chain loci are operably linked to the constant region thereof, and the gene segments in transgenic light chain loci are operably linked to the constant region thereof, so that upon immunisation the vertebrate is capable of producing an antibody comprising heavy chains produced by recombination of a heavy chain locus and light chains produced by recombination of a light chain locus, wherein the heavy and light chains comprise human variable regions;
  • the population comprises (i) a first vertebrate type (lambda vertebrates) wherein said light chain loci comprise one or more human V ⁇ gene segments
  • the vertebrates have been immunised with the same antigen; optionally a human antigen.
  • immunisation of the lambda vertebrates is separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day from the immunisation of the kappa vertebrates.
  • the lambda vertebrates express substantially no kappa-type antibodies.
  • endogenous kappa antibody expression is substantially inactive in the lambda vertebrates.
  • the kappa vertebrates express substantially no lambda-type antibodies.
  • endogenous lambda antibody expression is substantially inactive in the kappa vertebrates.
  • the invention provides an animal house or a laboratory containing a population according to the invention.
  • vertebrates of the population can be housed in the same cage or in the same collection of cages in the same animal house, building or laboratory.
  • the cages or vertebrates themselves may be labelled so that they are part of the same population or experiment. They may be owned by the same owner, eg, the same company, or in the control of a single person or company. They may be allocated for use in the same research programme or series of related research experiments aimed at discovering one or more antibodies or antibody chains against a common antigen or related antigens.
  • the vertebrates provide a population and are used as such.
  • vertebrates are discussed in the context of the same research programme or immunisation schedule or experiment or set of experiments in a laboratory notebook or a set of laboratory notebooks that relate to the same research programme or immunisation schedule or experiment or set of experiments.
  • a programme, schedule or experiment(s) may relate to immunisation of the vertebrates of a population with the same antigen.
  • the invention provides a selected repertoire of antibodies produced according to the method of the invention, or a second repertoire of antibodies produced according to the method of the invention, wherein the antibodies of the selected or second repertoire have been selected for binding a common antigen (eg, a human antigen) from a population of non-human vertebrates.
  • a common antigen eg, a human antigen
  • the vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related; optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the population is according to any configuration, aspect, embodiment, example or description of the invention herein.
  • the invention provides a non-human vertebrate (optionally a mouse or rat), wherein the genome of each vertebrate comprises:
  • transgenic immunoglobulin heavy chain loci each locus comprising one or more human V gene segments, one or more human D gene segments and one or more human J gene segments upstream of one or more heavy chain constant regions; and
  • transgenic immunoglobulin light chain loci eg, lambda loci or kappa loci
  • each locus comprising a human V ⁇ gene segment repertoire and one or more human J ⁇ gene segments upstream of one or more light chain constant regions
  • the light chain loci express light chain sequences comprising variable region sequences derived from human V ⁇ gene segments (human lambda light chain sequences);
  • the kappa (and optionally endogenous lambda) light chain expression has been substantially inactivated so that the vertebrate expresses more human lambda light chain sequences than kappa light chain sequences (sequences of light chains comprising variable region sequences derived from V ⁇ gene segments);
  • endogenous heavy chain expression has been substantially inactivated;
  • Such vertebrates express light chains wherein all or substantially all light chains comprise a human lambda light chain sequence and the vertebrates express substantially no endogenous Ig chains or kappa chains.
  • Such vertebrates are useful as a population of lambda vertebrates for use in the seventh and tenth configuration of the invention (ie, embodiments of the invention where there are separate lambda vertebrates and kappa vertebrates).
  • kappa antibodies For example, less than 10, 5, 4, 3, 2, 1 or 0.5% of kappa antibodies are endogenous or no kappa antibodies are endogenous.
  • lambda antibodies For example, less than 10, 5, 4, 3, 2, 1 or 0.5% of lambda antibodies are endogenous or no lambda antibodies are endogenous. Due to the relatively low endogenous lambda expression in mice, when the vertebrates are mice, it may not be necessary to carry out any specific genetic manipulation in the mice to achieve substantially inactive lambda expression.
  • heavy antibodies are endogenous or no heavy antibodies are endogenous.
  • each said transgenic light chain locus comprises at least 20, 25, 26, 27, 28 or 29 human V ⁇ gene segments selected from the group consisting of V3-1, V2-8, V3-9, V3-10, V2-11, V3-12, V3-16, V2-18, V3-19, V3-21, V3-22, V2-23, V3-25, V3-27, V1-36, V5-37, V5-39, V1-40, V7-43, V1-44, V5-45, V7-46, V1-74, V9-49, V1-51, V5-52, V6-57, V4-60, V8-61 and V4-69; optionally all of the gene segments of the group.
  • each said transgenic light chain locus comprises a substantially complete functional J ⁇ gene segment repertoire of a human.
  • each said transgenic light chain locus comprises at least 3 or 4 human J ⁇ gene segments selected from the group consisting of J1, J2, J3, J6 and J7; optionally all of the gene segments of the group.
  • each said transgenic heavy chain locus comprises a substantially complete functional VH gene segment repertoire of a human.
  • each said transgenic heavy chain locus comprises at least 30, 35, 36, 37, 38 or 39 human VH gene segments selected from the group consisting of V6-1, V1-2, V1-3, V4-4, V7-41, V2-5, V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46, V3-48, V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74; optionally all of the gene segments of the group.
  • each said transgenic heavy chain locus comprises at least 4 or 5 human JH gene segments selected from the group consisting of J1, J2, J3, J4, J5, J6; optionally all of the gene segments of the group.
  • each said transgenic heavy chain locus comprises at least 15, 20, 21, 22, 23, 24, 25 or 26 human D gene segments selected from the group consisting of D1-1, D2-2, D3-3, D4-4, D5-5, D6-6, D1-7, D2-8, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D1-26, D6-25 and D7-27; optionally all of the gene segments of the group.
  • the invention provides a population of non-human vertebrates (optionally mice or rats), wherein each vertebrate is according to the invention, wherein the population provides a human VH gene segment repertoire that is divided between two or more vertebrates of said population as per the invention.
  • the invention provides a population of non-human vertebrates (optionally mice or rats), wherein each vertebrate is according to the invention, wherein the population provides a human VH gene segment repertoire that is divided between first, second and third vertebrates, wherein
  • the first vertebrate comprises a human VH gene repertoire comprising 5 or 6 gene segments from the group consisting of V6-1, V1-2, V1-3, V4-4, V7-41 and V2-5;
  • the second vertebrate comprises a human VH gene repertoire comprising 15, 16, 17, 18, 19, 20 or 21 gene segments from the group consisting of V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48;
  • the third vertebrate comprises a human VH gene repertoire comprising 10, 11, 12 or 13 gene segments from the group consisting of V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74; Wherein
  • the first vertebrate comprises a human VH gene repertoire consisting of V6-1, V1-2, V1-3, V4-4, V7-41 and V2-5;
  • the second vertebrate comprises a human VH gene repertoire consisting of V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48;
  • the third vertebrate comprises a human VH gene repertoire consisting of V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74.
  • the vertebrates express no kappa light chains at all.
  • the invention provides a method of isolating an antibody (lambda-type antibody) that binds a predetermined antigen and whose heavy and light chain variable regions are derived from human VH and V ⁇ gene segments respectively, the method comprising immunising the vertebrate or the population of vertebrates according to the invention with the antigen and selecting a lambda-type antibody from said vertebrate or population.
  • an antibody lambda-type antibody
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody selected as described above or a derivative thereof that binds said antigen, together with a pharmaceutically acceptable diluent, carrier or excipient.
  • derivative antibodies are antibodies that have one or more mutations compared to the isolated antibody (eg, to improve antigen-binding affinity and/or to enhance or inactivate Fc function) Such mutants specifically bind the antigen.
  • Mutation or adaptation to produce a derivative includes, eg, mutation to produce Fc enhancement or inactivation.
  • a derivative can be an antibody following conjugation to a toxic payload or reporter or label or other active moiety.
  • a chimaeric antibody chain or antibody is modified by replacing one or all human constant regions thereof by a corresponding human constant region.
  • the invention provides a method of providing a synthetic antibody heavy chain sequence repertoire, the method comprising providing a heavy chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous heavy chain expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic heavy chain loci comprising one or more VH gene segments, one or more D gene segments and one or JH gene segments functionally connected upstream of a heavy chain constant region (eg, Cmu and/or Cgamma), wherein the genomes can express different repertoires of antibody heavy chain sequences derived from VH, D and JH gene segments;
  • a heavy chain constant region eg, Cmu and/or Cgamma
  • the gene segment repertoire is selected from the group consisting of: (a) a VH gene repertoire (eg, a human VH gene repertoire or a substantially complete functional human VH gene repertoire); (b) a D gene repertoire (eg, a human D gene repertoire or a substantially complete functional human D gene repertoire); and (c) a JH gene repertoire (eg, a human JH gene repertoire or a substantially complete functional human JH gene repertoire); Optionally wherein the D and JH segments in the loci are human D and JH segments.
  • a VH gene repertoire eg, a human VH gene repertoire or a substantially complete functional human VH gene repertoire
  • D gene repertoire eg, a human D gene repertoire or a substantially complete functional human D gene repertoire
  • a JH gene repertoire eg, a human JH gene repertoire or a substantially complete functional human JH gene repertoire
  • the invention provides a method of providing a synthetic antibody kappa chain sequence repertoire, the method comprising providing a kappa chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic light chain loci comprising one or more V ⁇ gene segments and one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody kappa chain sequences derived from V ⁇ and J ⁇ gene segments;
  • a light chain constant region eg, C ⁇ and/or C ⁇
  • the gene segment repertoire is selected from the group consisting of: (a) a V ⁇ gene repertoire (eg, a human V ⁇ gene repertoire or a substantially complete functional human V ⁇ gene repertoire); and (c) a J ⁇ gene repertoire (eg, a human J ⁇ gene repertoire or a substantially complete functional human J ⁇ gene repertoire); Optionally wherein the J ⁇ segments in the loci are human J ⁇ segments.
  • the invention provides a method of providing a synthetic antibody lambda chain sequence repertoire, the method comprising providing a lambda chain variable region gene segment repertoire that is divided across the genomes of two or more non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive, the repertoire gene segments in the genomes being provided as part of transgenic light chain loci comprising one or more V ⁇ gene segments and one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ ), wherein the genomes can express different repertoires of antibody lambda chain sequences derived from V ⁇ and J ⁇ gene segments;
  • a light chain constant region eg, C ⁇ and/or C ⁇
  • the gene segment repertoire is selected from the group consisting of: (a) a V ⁇ gene repertoire (eg, a human V ⁇ gene repertoire or a substantially complete functional human V ⁇ gene repertoire); and (c) a J ⁇ gene repertoire (eg, a human J ⁇ gene repertoire or a substantially complete functional human J ⁇ gene repertoire); Optionally wherein the J ⁇ segments in the loci are human J ⁇ segments.
  • the invention provides a method of providing a synthetic antibody light chain sequence repertoire, the method comprising providing
  • V ⁇ gene repertoire in the genomes of a first group of non-human vertebrates in which endogenous lambda chain (and optionally also endogenous kappa chain) expression is substantially inactive the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇ , eg, human C ⁇ ), wherein the genomes can express repertoires of antibody kappa light chain sequences derived from V ⁇ and J ⁇ gene segments substantially in the absence of lambda light chain expression; and a V ⁇ gene repertoire in the genomes of a second group of non-human vertebrates in which endogenous kappa chain (and optionally also endogenous lambda chain) expression is substantially inactive, the V ⁇ genes in the genomes being provided as part of transgenic light chain loci comprising one or more J ⁇ gene segments functionally connected upstream of a light chain constant region (eg, C ⁇ and/or C ⁇
  • the invention provides a population of non-human vertebrates for generating antibodies, the population comprising the first and second groups of vertebrates described above, optionally wherein the vertebrates of the population have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the invention provides a synthetic repertoire of antibody heavy chain sequences, antibody light chain sequences, antibody kappa chain sequences, antibody lambda chain sequences, or a repertoire of antibodies obtained from a population of non-human vertebrates according to the invention; or wherein the repertoire is obtained from a vertebrate of the invention.
  • the population of vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the population of vertebrates are na ⁇ ve.
  • the vertebrates are na ⁇ ve (ie, not immunised with a predetermined antigen, as the term is understood in the art; for example, such a vertebrate that has been kept in a relatively sterile environment as provided by an animal house used for R&D).
  • the vertebrates have been immunised with a predetermined antigen, eg, an antigen bearing a human epitope.
  • the population comprises na ⁇ ve and immunised vertebrates.
  • the genomes of the vertebrates comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction.
  • the members of the repertoire bind a common antigen and have been generated in the same research programme.
  • the invention provides a method of providing a synthetic antibody heavy chain repertoire, the method comprising
  • a human VH gene segment repertoire (eg, a substantially complete functional human VH gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of VH gene segments corresponding to a distal VH gene cluster of the heavy chain locus of a human; and a second cluster of VH gene segments corresponding to a proximal VH gene cluster of the heavy chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a heavy chain locus of said first vertebrate upstream of one or more D gene segments, one or more JH gene segments and one or more constant regions; Wherein the proximal cluster is provided in a heavy chain locus of said second vertebrate upstream of one or more D gene segments, one or more JH gene segments and one or more constant regions; Wherein the proximal VH gene cluster is not present between
  • a cluster of human gene segments refers to a collection of gene segments present in a human Ig locus (eg, a human heavy chain locus) from a first gene segment to a second, downstream (more 3′) gene segment in a human genome (eg, in a germline human Ig locus). Examples of gene segment arrangements are found in the IMGT database (or by reference to Kabat or the other antibody resources available to the skilled person and described herein).
  • the gene segments in a cluster are all human VH.
  • the gene segments in a cluster are all human V ⁇ gene segments.
  • the gene segments in a cluster are all human V ⁇ gene segments.
  • the gene segments in a cluster are all human JH gene segments.
  • the gene segments in a cluster are all human J ⁇ gene segments.
  • the gene segments in a cluster are all human J ⁇ gene segments.
  • the gene segments in a cluster are all human D gene segments.
  • a human germline heavy chain Ig locus comprises the following gene cluster (in the 3′ to 5′ direction, ie, in the proximal to distal direction, ie in the downstream to upstream direction): V6-1, V1-2, V1-3, V4-4, V7-41.
  • the V gene segments in this cluster can be provided in this order (the human germline order) or a different order from that shown here.
  • a “distal cluster” could be (in the 3′ to 5′ direction, ie, in the proximal to distal direction, ie in the downstream to upstream direction): V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74.
  • a medial cluster could be (in the 3′ to 5′ direction, ie, in the proximal to distal direction, ie in the downstream to upstream direction): V3-7, V1-8, V3-9, V3-11, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48.
  • the gene segment clusters are discrete (ie, do not share a common gene segment).
  • a first gene segment cluster (such as a proximal cluster) comprises one or more gene segments that are comprised by a second gene segment cluster (such as a medial or distal cluster) and the first and second clusters are different.
  • a third vertebrate comprises a third cluster of VH gene segments corresponding to a medial VH gene cluster of the heavy chain locus of a human, wherein the medial cluster is arranged between the distal and proximal clusters in said human locus; wherein the medial cluster is provided in a heavy chain locus of said third vertebrate upstream of one or more D gene segments, one or more JH gene segments and one or more constant regions; wherein the heavy chain locus of the third vertebrate does not comprise the distal, medial and proximal clusters in an arrangement corresponding to the arrangement of said human (optionally wherein the heavy chain locus of the third vertebrate does not comprise the distal and proximal clusters); the method further comprising expressing said heavy chain locus of the third vertebrate, whereby the repertoire of synthetic antibody heavy chains is provided by expression from the first, second and third vertebrate loci.
  • the vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the vertebrates are na ⁇ ve.
  • the vertebrates have a common collection of light chain loci.
  • the kappa chain loci alleles are identical in the vertebrates and/or the lambda chain loci alleles are identical in the vertebrates. This simplifies construction of vertebrate variants for producing the population and also simplifies breeding.
  • the method comprises comprising selecting one or more antibody heavy chains (eg, as part of an antibodies) from said repertoire according to a desired characteristic (eg, affinity for biding an antigen).
  • a desired characteristic eg, affinity for biding an antigen
  • the invention provides a method of providing a synthetic antibody kappa chain repertoire, the method comprising
  • a human V ⁇ gene segment repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of V ⁇ gene segments corresponding to a distal V ⁇ gene cluster of the kappa chain locus of a human; and a second cluster of V ⁇ gene segments corresponding to a proximal V ⁇ gene cluster of the kappa chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a kappa chain locus of said first vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal cluster is provided in a kappa chain locus of said second vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal V ⁇ gene cluster is not present between the distal cluster and a
  • a third vertebrate comprises a third cluster of V ⁇ gene segments corresponding to a medial V ⁇ gene cluster of the kappa chain locus of a human, wherein the medial cluster is arranged between the distal and proximal clusters in said human locus; wherein the medial cluster is provided in a kappa chain locus of said third vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; wherein the kappa chain locus of the third vertebrate does not comprise the distal, medial and proximal clusters in an arrangement corresponding to the arrangement of said human (optionally wherein the kappa chain locus of the third vertebrate does not comprise the distal and proximal clusters); the method further comprising expressing said kappa chain locus of the third vertebrate, whereby the repertoire of synthetic antibody kappa chains is provided by expression from the first, second and third vertebrate loci.
  • the invention provides a method of providing a synthetic antibody lambda chain repertoire, the method comprising
  • a human V ⁇ gene segment repertoire (eg, a substantially complete functional human V ⁇ gene repertoire) across the genomes of at least first and second non-human vertebrates (eg, mice or rats), the repertoire comprising a first cluster of V ⁇ gene segments corresponding to a distal V ⁇ gene cluster of the lambda chain locus of a human; and a second cluster of V ⁇ gene segments corresponding to a proximal V ⁇ gene cluster of the lambda chain locus of a human, wherein the proximal cluster is arranged proximally to the distal cluster in said human locus; Wherein the distal cluster is provided in a lambda chain locus of said first vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal cluster is provided in a lambda chain locus of said second vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; Wherein the proximal V ⁇ gene cluster is not present between the distal cluster and the
  • a third vertebrate comprises a third cluster of V ⁇ gene segments corresponding to a medial V ⁇ gene cluster of the lambda chain locus of a human, wherein the medial cluster is arranged between the distal and proximal clusters in said human locus; wherein the medial cluster is provided in a lambda chain locus of said third vertebrate upstream of one or more J ⁇ gene segments and one or more constant regions; wherein the lambda chain locus of the third vertebrate does not comprise the distal, medial and proximal clusters in an arrangement corresponding to the arrangement of said human (optionally wherein the lambda chain locus of the third vertebrate does not comprise the distal and proximal clusters); the method further comprising expressing said lambda chain locus of the third vertebrate, whereby the repertoire of synthetic antibody lambda chains is provided by expression from the first, second and third vertebrate loci.
  • the vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the vertebrates are na ⁇ ve.
  • the vertebrates have a common collection of heavy chain loci.
  • the method comprises selecting one or more antibody kappa or lambda chains (eg, as part of an antibodies) from said repertoire according to a desired characteristic (eg, affinity for biding an antigen).
  • a desired characteristic eg, affinity for biding an antigen
  • the inventors carried out sectoring using a repertoire in which they inverted human gene segments that are naturally in an opposite 5′-3′ orientation in a human germline genome (eg, see the Ig kappa locus arrangement, eg, as shown in the IMGT database and Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 161-174 (2001)—see 5′ gene segments marked with “D”).
  • the distribution of human gene segment usage in transgenic non-human vertebrates surprisingly demonstrated that artificially inverted gene segments were used for rearrangement and expression, the usage unexpectedly being relatively high ( FIG. 10 ).
  • the invention provides a non-human vertebrate (optionally a mouse or a rat) or vertebrate cell (optionally a mouse cell or a rat cell) whose genome comprises a transgenic antibody chain locus comprising one or more human V gene segments and one or more human J gene segments (and optionally one or more human D gene segments) upstream of a constant region, the locus comprising one or more inverted vertebrate species gene segments, the inverted gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the vertebrate species germline orientation of such segment(s), and wherein the non-human vertebrate or cell is capable of expressing an antibody chain sequence comprising a sequence that is derived from an inverted gene segment.
  • the cell is a B-cell, hybridoma, ES cell or iPS cell.
  • ES cells and iPS cells can be used to develop corresponding non-human vertebrates (vertebrates of the invention) in which the inverted gene segment(s) are functional as the inventors surprisingly observed.
  • the inverted gene segment(s) are V gene segments.
  • the vertebrate species is selected from human, mouse, rat, rabbit, guinea pig, chicken, a fish, a bird, a reptile, a Camelid, bovine, chimpanzee, a non-human primate and a primate.
  • the vertebrate species is human and optionally also the vertebrate of the invention is a mouse or rat.
  • the invention also provides a non-human vertebrate (optionally a mouse or a rat) or vertebrate cell (optionally a mouse cell or a rat cell) whose genome comprises a transgenic antibody chain locus comprising one or more human V gene segments and one or more human J gene segments (and optionally one or more human D gene segments) upstream of a constant region, wherein the locus comprises one or more inverted human gene segments, the inverted human gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the human germline orientation of such segment(s), and wherein the vertebrate or cell is capable of expressing an antibody chain sequence comprising a variable region that is derived from recombination of an inverted gene segment.
  • the inverted gene segment(s) are or comprise human V ⁇ gene segment(s).
  • the inverted V ⁇ gene segment(s) comprise one or more or all of V ⁇ 2D-40, 1D-39, 1D-33, 2D-30, 2D-29, 2D-28, 2D-26, 3D-20, 1D-17, 1D-16, 1D-13, 1D-12, 3D-11, 1D-43, 1D-8 and 3D-7 and optionally also 3D-15.
  • the locus comprises human V ⁇ gene segments comprising V ⁇ 4-1 and/or 5-2 with one or more or all of V ⁇ 2D-40, 1D-39, 1D-33, 2D-30, 2D-29, 2D-28, 2D-26, 3D-20, 1D-17, 1D-16, 1D-13, 1D-12, 3D-11, 1D-43, 1D-8 and 3D-7, and optionally also 3D-15, wherein all of the human V ⁇ gene segments are in the same orientation as the constant region of the locus.
  • the locus comprises one, more or all of human V ⁇ 2D-40, 1D-39, 1D-33 and 2D-30 3′ of one more or all of V ⁇ 3D-7, 1D-8, 1D-43 and 3D-11.
  • V ⁇ 2D-40, 1D-39, 1D-33 and 2D-30 are present in this order 3′ to 5′ in the locus.
  • V ⁇ 3D-7, 1D-8, 1D-43 and 3D-11 are present in this order 3′ to 5′ in the locus.
  • the 5′-most inverted V ⁇ gene segment in the locus is V ⁇ 3D-20. In another example, it is V ⁇ 2D-40 (eg, if the sequence of naturally-inverted human V ⁇ gene segments found in a wild-type human chromosome 2 are provided as this sequence, but in reverse orientation—with V ⁇ 2D-40 as the 5′-most inverted gene segment in the locus of the invention and V ⁇ 3D-7 is the 3′-most inverted gene segment).
  • the V ⁇ gene segment(s) comprise human V ⁇ 2D-40.
  • the V ⁇ gene segment(s) comprise human V ⁇ 1D-39, for example human V ⁇ 2D-40 next to human V ⁇ 1D-39 (eg, in 5′ to 3′ direction: human V ⁇ 2D-40 next to human V ⁇ 1D-39).
  • the constant region is human.
  • the constant region is a mouse or rat constant region, eg, a constant region endogenous to the non-human vertebrate.
  • the invention also provides method of providing an artificial human antibody variable region repertoire, the method comprising inserting one or more human V gene segment(s) (inverted gene segments) upstream of one or more J gene segments, optionally one or more D gene segments, and a constant region in an antibody chain locus of a non-human vertebrate or non-human vertebrate cell, the V gene segment(s) being present in the locus in a 5′-3′ orientation that is opposite to the human germline orientation of such segment(s), and wherein the non-human vertebrate or cell (or a non-human vertebrate progeny derived from the cell) is capable of expressing an antibody chain sequence comprising a variable region sequence that is derived from recombination of an inverted gene segment.
  • vertebrate or cell or inverted gene segment(s) is as recited above.
  • the invention also provides a method of providing an artificial human antibody variable region repertoire, the method comprising isolating serum or a lymphoid cell (eg, spleen cell or B-cell) from a vertebrate of the invention, and optionally isolating from the serum or cell one or more antibodies that specifically bind a predetermined antigen.
  • a lymphoid cell eg, spleen cell or B-cell
  • the invention also provides an antibody isolated in the method described above, or a fragment or derivative thereof.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps
  • compositions, populations, vertebrates, antibodies, repertoires and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • the inventors realised that the prior art mice provide for immunoglobulin loci in which all of the desired heavy, kappa and lambda gene segment repertoires are provided together in the genome of the cells of the individual mice.
  • the prior art designs have not addressed the limitation of the restricted accessible B-cell component size (around 2 ⁇ 10 8 B-cells in a mouse, for example) and the concomitant restriction on the overall antibody chain diversity that can be expressed and accessed in any one mouse.
  • FIG. 1 shows a calculation of potential diversity for a transgenic heavy chain locus comprising a complete functional repertoire of human VH, D and JH gene segments (in this example, 6 different human JH, 27 different human D and 40 different human VH germline gene segments).
  • the calculation takes into account junctional diversity (typically a 3 nucleotide addition at the J-D junction and a 2 nucleotide addition at the D-V junction).
  • junctional diversity typically a 3 nucleotide addition at the J-D junction and a 2 nucleotide addition at the D-V junction.
  • the calculation of potential diversity of heavy chain variable region sequences produced by recombination of the gene segments is 4 ⁇ 10 10 .
  • FIG. 2 shows a calculation of potential diversity for a transgenic kappa and lambda light chain loci, each comprising a complete functional repertoire of human VH, D and JH gene segments (in this example, 5 different human JL and 40 different human VL germline gene segments in each light chain locus).
  • the calculation takes into account junctional diversity (typically a 1 nucleotide addition at the V-J junction).
  • junctional diversity typically a 1 nucleotide addition at the V-J junction.
  • the calculation of potential diversity of kappa and lambda chain variable region sequences produced by recombination of the gene segments is 1 ⁇ 10 4 .
  • FIG. 3 shows a calculation of potential diversity for transgenic heavy, kappa and lambda light chain loci (ie, the calculation of total potential heavy/light chain variable region combinations).
  • the calculated result is a potential diversity of 4 ⁇ 10 14 .
  • FIG. 4 schematically illustrates the disadvantage of the existing theoretical designs where mice will comprise human heavy, kappa and lambda gene repertoires together in the same mouse.
  • the state of the art has not reported the successful production of a mouse containing complete functional human heavy, kappa and lambda gene segment repertoires altogether in one mouse genome. Nevertheless, if the skilled person did do this (by, for example, using the complete repertoires described in FIGS. 1-3 ) from a possible antibody variable region repertoire size of 4 ⁇ 10 14 only a maximum of around 2 ⁇ 10 8 antibodies can be sampled from the accessible B-cell compartment (actually the art often struggles even to sample at anywhere near this level). Thus, a huge amount of the potential diversity cannot be sampled.
  • the present inventors have addressed this problem by sectoring the overall desired repertoire in transgenic animals so that the repertoire is instead accessed not in a single genome, but across a plurality of genomes making up a population.
  • This is schematically illustrated in FIG. 5 for an example where the light chain repertoire is sectored to separate lambda gene segment diversity from kappa gene segment diversity. This is possible, for example, by the provision of separate lambda vertebrates and kappa vertebrates according to the invention.
  • all mice comprise the same transgenic heavy chain locus (the locus illustrated in FIG. 1 in which there is a complete human functional diversity of V, D and J gene segments).
  • the complete functional human V ⁇ gene segment repertoire has been divided across three mice, so that the mice comprise different human V ⁇ gene segment sub-repertoires (in this case the sub-repertoires are not overlapping, although it is possible for them to overlap as long as they are not identical in their collections of gene segments).
  • a complete functional V ⁇ gene segment repertoire has been sectored across three different kappa vertebrates (right-hand side of the figure).
  • the large circle at the centre of the figure represents the potential overall diversity of 4 ⁇ 10 14 and each smaller circle represents the accessible B-cell compartment of the respective mice. It can be readily appreciated that this example allows for six individual samplings (sub-repertoires) of the overall potential sequence space.
  • these sub-repertoires together provide a population diversity in which the overall repertoire size is significantly greater than possible with the conventional design shown in FIG. 4 .
  • the repertoires in the lambda mice are novel, synthetic repertoires that are not seen in conventional transgenic mice that express prior art kappa and lambda antibody ratios together in the same mouse.
  • all antibodies in the lambda mice will comprise lambda light chains, and this is not seen in mice in nature.
  • the endogenous lambda expression is totally inactivated or negligible
  • the kappa mice kappa-type antibodies are essentially exclusively expressed.
  • the lambda-type and kappa-type repertoires of antibodies expressed in the lambda and kappa mice provide an overall antibody repertoire that is novel, usefully extended and synthetic (not seen in nature).
  • FIG. 6 shows a schematic example where the heavy chain VH gene segment repertoire is sectored according to the invention.
  • the repertoire is a complete human functional VH gene repertoire divided across three mice (mice “A”, “B” and “C”) as follows:—
  • V6-1 V1-2, V1-3, V4-4, V7-41, V2-5, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 & V3-48
  • V6-1 V1-2, V1-3, V4-4, V7-41, V2-5, V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 & V3-74
  • the V gene repertoires of mice A, B and C overlap, the repertoires are unique.
  • the repertoires do not overlap, eg, the VH gene repertoires are non-overlapping and comprise or consist of as follows:—
  • V3-49 V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74
  • VH gene repertoires comprise or consist of as follows:—
  • V4-28 V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48
  • the VH gene sub-repertoire of each mouse is provided in a transgenic heavy chain locus comprising a complete functional human JH gene repertoire and a complete functional human D gene repertoire. Endogenous heavy chain expression is inactivated. It is possible to provide each mouse with the same light chain loci (eg, one or more transgenic lambda locus and one or more transgenic kappa locus, wherein each light chain locus comprises human VL and JL gene segments). In the example of FIG. 6 , however, the lambda and kappa gene repertoires have also been sectored according to the invention as per the illustration in FIG. 5 . In either example, endogenous kappa chain expression is inactivated in the lambda mice (and endogenous lambda chain expression is optionally inactivated in the kappa mice).
  • the JH and D repertoire consists of or comprises
  • the JH and D repertoire consists of or comprises
  • VH, DH and JH gene repertoires comprise or consist of human gene segments as follows:—
  • V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48 (optionally wherein this represents the 5′ to 3′ order of the V gene segments)
  • first and second non-human vertebrates eg, mice
  • the heavy chain gene segment repertoires comprise identical repertoires of human VH gene segments and different human D gene segment repertoires
  • the first vertebrate D gene segment repertoire comprises D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D1-26 and D7-27;
  • the second vertebrate D gene segment repertoire comprises D1-1, D2-2, D3-3, D4-4, D5-5, D6-6, D1-7, D2-8, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D1-26 and D7-27; and optionally the vertebrates comprise identical JH gene repertoires (eg, J1, J2, J3, J4, J5 and J6).
  • each human VH gene segment repertoire comprises or consists of human VH gene segments (a) V6-1, V1-2, V1-3, V4-4, V7-41, V2-5, V3-7, V1-8, V3-9, V3-11, V3-13, V3-15, V1-18, V3-20, V3-21, V3-23, V1-24 and V2-26 and/or (b) V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, V1-45, V1-46 and V3-48 and/or (c) V3-49, V5-51, V3-53, V1-58, V4-59, V4-61, V3-64, V3-66, V1-69, V2-70, V3-72, V3-73 and V3-74.
  • each human VH gene segment repertoire comprises or consists of human VH gene segments (a), (b) and (c).
  • each mouse provides a unique sub-repertoire of antibodies and antibody heavy and light chain sequences.
  • the design enables the human V ⁇ diversity to be explored much more fully than present with prior art designs, due to the sectoring of the kappa and lambda repertoires as explained above.
  • sectoring of the human VH gene repertoire provides for new gene segment arrangements in the heavy chain loci (bringing distal gene segments more proximal and providing non-natural combinations of VH gene segments together in single loci). This enables exploration of the human VH gene diversity more fully as hitherto previously possible.
  • the unique VH gene repertoires are explored separately in mice in the context of lambda vertebrates separately from kappa vertebrates, which in turn provides for novel combinations not seen in nature or hitherto previously possible.
  • the transgenic mice shown in FIGS. 5 and 6 provide useful sources of antibody and antibody chain repertoires for sampling following immunisation with antigen.
  • all of the mice shown in the population of FIG. 5 or 6 can be immunised with the same human antigen and desirable antibodies can be selected, eg, for relatively high affinity binding to the antigen or for binding in certain epitopic regions of the antigen (eg, as determined by competition with an anti-antigen antibody of known epitopic specificity).
  • the selected antibodies thereby provide a selected repertoire of antibodies (optionally pooled) from which one or more preferred drug candidates can be selected.
  • This selection can be on the basis of the information already obtained in the first round of selection, and/or a further set of experiments (eg, ELISA and/or surface plasmon resonance (eg, using BiacoreTM) to determine affinities within the selected repertoire) can be performed to enable the selection from the repertoire.
  • a further set of experiments eg, ELISA and/or surface plasmon resonance (eg, using BiacoreTM) to determine affinities within the selected repertoire
  • a functional human gene segment repertoire (from V ⁇ 2D-40 to J ⁇ 5, as shown in FIG. 7 ) was sectored by the inventors to produce three different transgenic kappa chain alleles (denoted K1, K2 and K3) and corresponding mice.
  • the transgenic alleles were expressed in the mice and the kappa chain repertoires were assessed at the RNA transcript level.
  • Insertion of human kappa gene segments from a 1st IGK BAC into the IGK locus of mouse AB2.1 ES cells was performed to create a light chain allele denoted the K1 allele.
  • the inserted human sequence corresponds to the sequence of human chromosome 2 from position 89312220 to position 89159079 and comprises functional kappa gene segments V ⁇ 1-9, V ⁇ 1-8, V ⁇ 1-6, V ⁇ 1-5, V ⁇ 5-2, V ⁇ 4-1, J ⁇ 1, J ⁇ 2, J ⁇ 3, J ⁇ 4 and J ⁇ 5 ( FIG. 7 ).
  • the insertion was made between positions 70674755 and 70674756 on mouse chromosome 6, which is upstream of the mouse C ⁇ region.
  • the mouse V ⁇ and J ⁇ gene segments were retained in the locus, immediately upstream of (5′ of) the inserted human kappa DNA.
  • the mouse lambda loci were left intact.
  • K2 was constructed in which more human functional V ⁇ gene segments were inserted upstream (5′) of the 5′-most V ⁇ inserted in the K1 allele by the sequential insertion of human DNA from a second BAC.
  • the inserted human sequence from BAC2 corresponds to the sequence of human chromosome 2 from position 89478399 to position 89312221 and comprises functional kappa gene segments V ⁇ 2-24, V ⁇ 1-17, V ⁇ 1-16, V ⁇ 3-15, V ⁇ 1-13, V ⁇ 1-12, V ⁇ 3-11 ( FIG. 7 ).
  • K3 was constructed in which more human functional V ⁇ gene segments were inserted upstream (5′) of the 5′-most V ⁇ inserted in the K2 allele by the sequential insertion of human DNA from a third BAC.
  • the inserted sequence corresponds to the sequence of human chromosome 2 from position 89889512 to position 89902788 and from position 89609410 to position 89478400, and comprises functional kappa gene segments, V ⁇ 2D-40, V ⁇ 1D-39, V ⁇ 1-33, V ⁇ 2-30, V ⁇ 2-29, V ⁇ 2-28 and V ⁇ 1-27 ( FIG. 7 ).
  • mice bearing either the K1, K2 or K3 insertion into an endogenous kappa locus were generated from the ES cells using standard procedures.
  • the other endogenous kappa locus was inactivated in the mice by insertion of an inactivating sequence comprising neo R into the mouse J ⁇ -C ⁇ intron (to produce the “KA” allele).
  • Standard 5′-RACE was carried out to analyse RNA transcripts from the transgenic kappa light chain loci in the K1, K2 and K3 mice.
  • the K3 mice showed high usage of human V ⁇ gene segments, 92.8% of rearranged transcripts using human V ⁇ gene segments and only 7.2% of them using mouse V ⁇ gene segments ( FIG. 8 ). Compared to K1 and K2, K3 showed improved human V ⁇ usage (K1: 78%; K2: 83% and K3: 93%) ( FIG. 9 ).
  • the distribution of human V ⁇ usage from K1 to K3 mice also demonstrated that insertion of different repertoires of human V ⁇ gene segments changes the usage of human V ⁇ s ( FIG. 11 ). While not wishing to be bound by any particular theory, the inventors surmise that this is due to the varying competition among the V ⁇ s, which determine their relative usage. This was also surprisingly observed with the human J ⁇ usage. For example, the inventors surprisingly observed that with the 3rd BAC insertion, the J ⁇ 4 usage is increased ( FIG. 12 ). Thus, the human V ⁇ gene repertoire sectoring according to the invention not only altered the repertoire of expressed human V ⁇ gene segments, but also altered the expressed J ⁇ profile. Thus, the inventors surprisingly realised that they had discovered a way to provide differing repertoires of antibody chains by gene repertoire sectoring.
  • K1, K2 and K3 as a combined population is useful, the inventors realised, to produce a novel repertoire of antibody chains and antibodies that can be selected against a desired antigen.
  • the K1, K2 and K3 mice can be immunised with the same human antigen and anti-antigen antibodies from the totally of antibodies in the mice can be selected on the basis of affinity and/or target epitope recognition or another desirable feature.
  • the analysis of kappa light chain sequences revealing differential usage and thus differing light chain sequence repertoires could be produced by the gene sectoring technique.
  • the repertoires differed in their human V and J gene segment usage.
  • the proportions of particular human gene segments shared by mice could be altered in the expression profiles by the gene sectoring technique of the present invention.
  • the inserted human sequence corresponds to the sequence of human chromosome 14 from position 106494908 to position 106328951 and comprises functional heavy gene segments V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, J H 1, J H 2, J H 3, J H 4, J H 5 and J H 6 (in 5′ to 3′ order).
  • mice V H , D and J H gene segments were retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA.
  • a second allele, S2 was constructed in which more human functional V H gene segments were inserted upstream (5′) of the 5′-most V H inserted in the S1 allele by the sequential insertion of human DNA from a second BAC (BAC2).
  • the inserted human sequence from BAC2 corresponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7.
  • a third allele, S3 was constructed in which more human functional V H gene segments were inserted upstream (5′) of the 5′-most V H inserted in the S2 allele by the sequential insertion of human DNA from a third BAC (BAC3).
  • the inserted sequence corresponds to the sequence of human chromosome 14 from position 106759988 to position 106609301, and comprises functional heavy chain gene segments, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, and V H 3-15.
  • mice bearing either the S1, S2 or S3 insertion into an endogenous heavy chain locus were generated from the ES cells using standard procedures.
  • the other endogenous heavy chain locus was inactivated in the mice by insertion of an inactivating sequence comprising neo R into the mouse J H -C ⁇ intron (to produce the “HA” allele).
  • Standard 5′-RACE was carried out to analyse RNA transcripts from the transgenic heavy chain light chain loci in the S1, S2 and S3 mice.
  • the S3 mice showed high usage of human V H gene segments, 62% of rearranged transcripts using human V H gene segments and 38% of them using mouse V ⁇ gene segments ( FIG. 13 ).
  • Human V H usage was as follows—S1: 16%; S2: 64% and S3: 62% ( FIG. 13 ).
  • the S1, S2 and S3 mice can be immunised with the same human antigen and anti-antigen antibodies from the totally of antibodies in the mice can be selected on the basis of affinity and/or target epitope recognition or another desirable feature.
  • V6 a fourth heavy chain allele, denoted “V6”, in which more human functional V H gene segments were inserted upstream (5′) of the 5′-most V H inserted in the S1 allele by the sequential insertion of human DNA from a BAC (BAC6).
  • the inserted human sequence from BAC6 corresponds to the sequence of human chromosome 14 from position 107147078 to position 106995083 and comprises functional heavy chain gene segments V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49 ( FIG. 16 ).
  • S2 and V6 mice showed greater human VH gene segment usage than S1 mice.
  • the usage in S2 compared to V6 was, furthermore, different. See also FIG. 16 .
  • human heavy gene segments are inserted from a 1st IGH BAC into the IGH locus of mouse AB2.1 ES cells (Baylor College of Medicine) is performed to create a heavy chain allele denoted the S1 allele.
  • the insertion is made between positions 114666435 and 114666436 on mouse chromosome 12, which is upstream of the mouse Cu region.
  • the mouse V H , D and J H gene segments are retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA (and subsequently inverted to inactivate at a later stage).
  • a second allele, S2 is constructed in which more human functional V H gene segments are inserted upstream (5′) of the 5′-most V H inserted in the S1 allele by the sequential insertion of human DNA from a second BAC (BAC2).
  • the inserted human sequence from BAC2 corresponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7.
  • a third allele, S3 is constructed in which more human functional V H gene segments is inserted upstream (5′) of the 5′-most V H inserted in the S2 allele by the sequential insertion of human DNA from a third BAC (BAC3).
  • the inserted sequence corresponds to the sequence of human chromosome 14 from position 106759988 to position 106609301, and comprises functional heavy chain gene segments, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, and V H 3-15.
  • version 1 (denoted S3F) had a human gene segment repertoire V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, J H 1, J H 2, J H 3, J H 4, J H 5 and J H 6 (in 5′ to 3′ order).
  • version 2 (denoted S3FD) had a human gene segment repertoire V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2, V H 6-1, D1-1, D2-2, D3-3, D4-4, D5-5, D6-6, D1-7, D2-8 , D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, J H 1, J H 2, J H 3, J H 4, J H 5 and J H 6 (in 5′ to 3′ order).
  • the underlined segments do not appear in S3F.
  • mice bearing either the S3F or S3FD insertion into an endogenous heavy chain locus are generated from the ES cells using standard procedures.
  • the other endogenous heavy chain locus is inactivated in the mice by insertion of an inactivating sequence comprising neo R into the mouse J H -C ⁇ intron (to produce the “HA” allele).
  • Standard 5′-RACE is carried out to analyse RNA transcripts from the transgenic heavy chain light chain loci in the S3F and S3FD mice.
  • K4 mice were generated as follows by the insertion of further DNA from the V ⁇ gene cluster of human chromosome 2.
  • K4 was constructed in which more human functional V ⁇ gene segments were inserted upstream (5′) of the 5′-most V ⁇ inserted in the K3 allele by the sequential insertion of human DNA from a fourth BAC.
  • the inserted sequence corresponds to the sequence of human chromosome 2 from position [90062244] to position [90276666], and comprises functional kappa gene segments [V ⁇ 3D-20, V ⁇ 1D-17, V ⁇ 1D-16, V ⁇ 1D-13, V ⁇ 1D-12, V ⁇ 3D-11, V ⁇ 1D-43, V ⁇ 1D-8, V ⁇ 3D-7] ( FIG. 17 ).
  • V ⁇ s are in the distal cluster of V ⁇ segment and naturally in an opposite orientation to the J ⁇ exons in germline human chromosome 2 but were inverted to the proximal human J ⁇ s and the mouse C ⁇ in this transgenic allele.
  • the K4 mice showed high usage of human V ⁇ s, 94.5% of rearranged transcripts using human V ⁇ s and only 5.5% of them using mouse V ⁇ s ( FIG. 18 ).
  • K1 and K2 showed improved human V ⁇ usage to K1 and K2 (K1: 78%; K2: 83%), and similar usage to K3 (K3: 93%) ( FIG. 19 ).
  • All the V ⁇ s from the distal cluster are rarely used in human (Cox, J P L et al, “A directory of human germ-lime V ⁇ segments reveals a strong bias in their usage”, Eur. J. Immunol. 1994. 24: 827-836).

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